Transgenic plants having altered biomass composition

ABSTRACT

Methods and materials for modulating biomass composition in plants are disclosed. For example, nucleic acids encoding biomass composition-modulating polypeptides are disclosed as well as methods for using such nucleic acids to transform plant cells. Also disclosed are plants having altered biomass composition and plant products produced from plants having altered biomass composition.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of International ApplicationNo. PCT/US2011/057709, filed Oct. 25, 2011, which claims the benefit ofU.S. Provisional Application No. 61/407,280, filed Oct. 27, 2010. Thecontents of the foregoing applications are hereby incorporated byreference in their entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This application is a division of U.S. Non-Provisional application Ser.No. 15/717,773, filed Sep. 27, 2017 which is a division of U.S.Non-Provisional application Ser. No. 13/828,225, filed Mar. 14, 2013(now issued U.S. Pat. No. 9,828,608) which is a continuation-in-part ofInternational Application No. PCT/US2011/057709, filed Oct. 25, 2011,which claims the benefit of U.S. Provisional Application No. 61/407,280,filed Oct. 27, 2010. The contents of the foregoing applications arehereby incorporated by reference in their entireties.

TECHNICAL FIELD

This document relates to methods and materials involved in modulatingbiomass composition in plants. For example, this document providesplants having altered sucrose or conversion efficiency, as well asmaterials and methods for making plants and plant products havingaltered sucrose or conversion efficiency.

BACKGROUND

Plants store energy from sunlight in the form of chemical bonds thatcompose plants. The energy stored in plant materials can be converted toforms of energy such as heat, electricity and liquid fuels, dependingupon the plant material employed and the process applied to extractenergy from it. Other processes can produce chemical intermediates fromplant biomass that are useful in a variety of industrial processes, forinstance lactic acid, succinic acid, etc.

Plant materials have been used for millennia by humans to generate heatby direct combustion in air. For building and process heating purposes,this heat is typically used to generate steam, which is a moretransportable heat source used to heat buildings and public areas usingheat exchangers of various design. The production of steam may also beused to drive turbines, which transform heat energy into electricalenergy. These processes typically involve a simple, direct combustionprocess of the plant material alone, or a co-firing process with coal orother energy source.

Fuels such as ethanol can be produced from plant materials by a numberof different processes. For example, the sucrose in sugarcane can beextracted from the plant material and directly fermented to ethanolusing a microorganism, such as brewer's yeast. Brazil has converted asignificant portion of its transportation sector over to ethanol derivedfrom sugarcane, proving this can be done on a very large scale overbroad geography. As another example, the starch from corn can beprocessed using α-amylase and glucoamylase to liberate free glucose thatis subsequently fermented to ethanol. The US uses a significant portionof its corn crop to produce ethanol from starch. While these advancesare significant, the ability to increase the amount of liquidtransportation fuel obtained from plant material is limited andinsufficient to achieve federally mandated renewable energy targetsbecause only a small fraction of the solar energy captured andtransformed into chemical energy in plants is converted into biofuels inthese industrial processes.

Plant material can be used for the production of cellulosic biofuels bybiochemical processes employing enzymes and/or microorganisms or bythermochemical processes such as Biomass to Liquids (BtL) technologyusing high temperature and non-enzymatic catalysts. There are alsoexamples of hybrid thermochemical/biochemical processes. Biochemicalprocesses typically employ physical and chemical pretreatments, enzymes,and microorganisms to deconstruct the lignocellulose matrix of biomassin order to liberate the fermentable from cellulose, hemicellulose, andother cell wall carbohydrates, which are subsequently fermented toethanol by a microorganism. Currently, many different processing methodsare being developed for biofuel production that employ differentstrategies for pretreatment, enzyme cocktails, and microorganisms. Manyof these processes are focused on the production of ethanol, but butanoland other useful molecules (e.g., lactic acid, succinic acid,polyalkanoates, etc.) can also be produced in this type of process. Theconversion product molecule produced is usually defined by themicroorganisms selected for fermentation.

Thermochemical processes employ very high temperatures in a low oxygen(i.e., O₂) environment to completely degrade the organic constituents ofbiomass to syngas, largely composed of molecular hydrogen (H₂) andcarbon monoxide (CO) gas. These simple molecules are then re-formed intomore useful and valuable molecules (fuels or chemical intermediates)utilizing a Fischer-Tropsch process or other methods usually employing achemical catalyst of some sort. These processes are effective atproducing biofuels that are similar to current petrochemical-basedhydrocarbon fuels (i.e., gasoline, diesel, jet fuel), although otherbiofuel molecules can also be produced in these types of processes(i.e., ethanol, butanol, kerosene).

A variant form of thermochemical process uses pyrolysis (i.e., thermaldegradation in the complete absence of oxygen) to partially degrade theorganic constituents present in plant biomass to a chemicallyheterogeneous liquid bio-oil. This serves to increase the energy densityof the biomass to facilitate transport to centralized processingfacilities where the bio-oil is further processed to a desired productslate.

The economic viability of biomass conversion processes is significantlyimpacted by the composition of the plant material and its conversionefficiency to heat, electricity, biofuels or chemical intermediatesunder specific processing conditions. For biochemical processesproducing biofuels or other chemicals, the recalcitrance of thelignocellulose matrix of the biomass is a major factor in conversionefficiency.

SUMMARY

The present invention relates to methods of altering biomass compositionin plants and plants generated thereby. Plants having altered biomasscomposition are useful for agriculture, forage, horticulture, biomass toenergy conversion, paper production, plant product production, and otherindustries. For example, this document features dedicated energy cropssuch as Panicum virgatum L. (switchgrass), Miscanthus x gigantus(miscanthus), Sorghum sp., and Saccharum sp. (sugar cane) having alteredbiomass composition.

This document features a method of producing a plant. The methodincludes growing a plant cell comprising an exogenous nucleic acid. Theexogenous nucleic acid includes a regulatory region operably linked to anucleotide sequence encoding a polypeptide, where the HMM bit score ofthe amino acid sequence of the polypeptide is greater than about 65,based on the HMM of the amino acid sequences depicted in one of FIGS.1-12 . A plant produced from the plant cell has a difference in biomasscomposition compared to the corresponding composition of a control plantthat does not comprise the nucleic acid. The difference in biomasscomposition in the plant can be a difference in the sucrose content orconversion efficiency.

This document also features a method of producing a plant that includesgrowing a plant cell comprising an exogenous nucleic acid. The exogenousnucleic acid comprises a regulatory region operably linked to anucleotide sequence encoding a polypeptide having 80 percent or greatersequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 7, 8, 10, 12, 14, 15, 17, 18, 19, 20,21, 22, 24, 26, 28, 29, 30, 32, 34, 36, 37, 39, 41, 43, 45, 47, 49, 50,52, 53, 55, 57, 59, 61, 63, 65, 66, 68, 70, 71, 72, 73, 75, 77, 78, 79,81, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 100, 101, 102, 104, 105,107, 109, 111, 113, 115, 117, 118, 120, 122, 124, 126, 128, 130, 132,133, 135, 136, 138, 139, 141, 143, 145, 147, 148, 149, 151, 152, 153,155, 157, 158, 160, 161, 162, 163, 164, 165, 167, 168, 170, 171, 172,173, 175, 177, 179, 181, 182, 184, 185, 187, 189, 190, 192, 194, 196,197, 199, 201, 202, 204, 205, 207, 208, 210, 211, 213, 215, 216, 218,220, 221, 222, 223, 225, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 248, 250, 251, 252, 254, 256, 258, 260, 261, 262, 264, 266,268, 270, 272, 274, 275, 276, 278, 280, 282, 283, 284, 285, 286, 288,289, 290, 292, 294, 295, 296, 297, 298, 299, 300, 302, 303, 305, 306,308, 309, 310, 311, 312, 314, 316, 317, 318, 320, 321, 323, 325, 326,327, 328, 329, 331, 333, 335, 336, 337, 338, 339, 340, 342, 344, 346,348, 350, 351, 353, 355, 357, 359, 360, 361, 362, 363, 364, 365, 366,368, 370, 372, 373, 374, 375, 376, 377, 379, 380, 381, 383, 384, 386,388, 390, 392, 393, 394, 396, 398, 400, 402, 404, 406, 407, 408, 410,412, 413, 414, 416, 418, 419, 420, 422, 423, 424, 426, 427, 429, 430,431, 433, 434, 436, 437, 439, 441, 442, 444, 446, 448, 449, 450, 451,452, 454, 456, 458, 459, 460, 462, 463, 465, 466, 468, 470, 472, 473,474, 476, 478, 479, 480, 481, 483, 485, 486, 488, 490, 492, 493, 495,497, 498, 500, 501, 503, 504, 506, 508, 509, 510, 512, 514, 515, 516,517, 519, 521, 522, 523, 525, 527, 529, 531, 533, 534, 535, 537, 539,541, 543, 545, 547, 549, 551, 552, 554, 556, 557, 559, 562, 564, 565,567, 568, 570, 572, 573, 574, 575, 576, 578, 580, 582, 584, 586, 588,589, 590, 592, 594, 596, 598, 599, 601, 602, 603, 605, 607, 608, 609,611, 613, 615, 617, 619, 621, 622, 624, 625, 627, 629, 630, 632, 634,636, 638, 641, 643, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654,656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 677, 679, 680,682, 684, 686, 687, 688, 689, 690, 691, 692, 694, 695, 697, 699, 701,702, 704, 706, 708, 710, 712, 713, 715, 716, 718, 720, 721, 723, 724,726, 727, 729, 730, 732, 733, 735, 736, 737, 739, 740, 742, 744, 746,747, 748, 749, 750, 751, 753, 755, 757, 758, 760, 761, 763, 764, 765,766, 767, 768, 769, 771, 772, 774, 775, 776, 777, 778, 780, 781, 782,783, 785, 786, 788, 789, 791, 793, 795, 796, 798, 799, 800, 801, 802,803, 805, 807, 808, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819,820, 821, and 823. A plant produced from the plant cell has a differencein biomass composition as compared to the corresponding composition of acontrol plant that does not comprise the nucleic acid. The difference inbiomass composition in the plant can be a difference in the sucrosecontent or conversion efficiency.

In another aspect, this document features a method of producing a plant.The method includes growing a plant cell comprising an exogenous nucleicacid, where the exogenous nucleic acid includes a regulatory regionoperably linked to a nucleotide sequence having 80 percent or greatersequence identity to a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, 3, 5, 9, 11, 13, 16, 23, 25, 27, 31, 33, 35,38, 40, 42, 44, 46, 48, 51, 54, 56, 58, 60, 62, 64, 67, 69, 74, 76, 80,83, 85, 87, 89, 91, 93, 95, 98, 103, 106, 108, 110, 112, 114, 116, 119,121, 123, 125, 127, 129, 131, 134, 137, 140, 142, 144, 146, 150, 154,156, 159, 166, 169, 174, 176, 178, 180, 183, 186, 188, 191, 193, 195,198, 200, 203, 206, 209, 212, 214, 217, 219, 224, 227, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 253, 255, 257, 259, 263, 265,267, 269, 271, 273, 277, 279, 281, 287, 291, 293, 301, 304, 307, 313,315, 319, 322, 324, 330, 332, 334, 341, 343, 345, 347, 349, 352, 354,356, 358, 367, 369, 371, 378, 382, 385, 387, 389, 391, 395, 397, 399,401, 403, 405, 409, 411, 415, 417, 421, 425, 428, 432, 435, 438, 440,443, 445, 447, 453, 455, 457, 461, 464, 467, 469, 471, 475, 477, 482,484, 487, 489, 491, 494, 496, 499, 502, 505, 507, 511, 513, 518, 520,524, 526, 528, 530, 532, 536, 538, 540, 542, 544, 546, 548, 550, 553,555, 558, 560, 561, 563, 566, 569, 571, 577, 579, 581, 583, 585, 587,591, 593, 595, 597, 600, 604, 606, 610, 612, 614, 616, 618, 620, 623,626, 628, 631, 633, 635, 637, 639, 640, 642, 644, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 678, 681, 683, 685, 693, 696, 698,700, 703, 705, 707, 709, 711, 714, 717, 719, 722, 725, 728, 731, 734,738, 741, 743, 745, 752, 754, 756, 759, 762, 770, 773, 779, 784, 787,790, 792, 794, 797, 804, 806, 809, and 822, or a fragment thereof. Aplant produced from the plant cell has a difference in biomasscomposition as compared to the corresponding composition of a controlplant that does not include the nucleic acid. The difference in biomasscomposition in the plant can be a difference in the sucrose content orconversion efficiency.

This document also features a method of producing a plant that includesgrowing a plant cell comprising an exogenous nucleic acid. The exogenousnucleic acid is effective for down regulating an endogenous nucleic acidin the plant cell, wherein the endogenous nucleic acid encodes apolypeptide, and wherein the HMM bit score of the amino acid sequence ofthe polypeptide is greater than about 65, where the HMM is based on theamino acid sequences depicted in one of FIGS. 1-12 .

In another aspect, this document features a method of modulating biomasscomposition in a plant. The method includes introducing into a plantcell an exogenous nucleic acid, the exogenous nucleic acid comprising aregulatory region operably linked to a nucleotide sequence encoding apolypeptide, wherein the HMM bit score of the amino acid sequence of thepolypeptide is greater than about 65, where the HMM is based on theamino acid sequences depicted in one of FIGS. 1-12 , and wherein a plantproduced from the plant cell has a difference in biomass composition ascompared to the corresponding composition of a control plant that doesnot comprise the exogenous nucleic acid. The difference in biomasscomposition in the plant can be a difference in the sucrose content orconversion efficiency.

A method of modulating biomass composition in a plant also is featured.The method includes introducing into a plant cell an exogenous nucleicacid, the exogenous nucleic acid comprising a regulatory region operablylinked to a nucleotide sequence encoding a polypeptide having 80 percentor greater sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2, 4, 6, 7, 8, 10, 12, 14, 15, 17, 18,19, 20, 21, 22, 24, 26, 28, 29, 30, 32, 34, 36, 37, 39, 41, 43, 45, 47,49, 50, 52, 53, 55, 57, 59, 61, 63, 65, 66, 68, 70, 71, 72, 73, 75, 77,78, 79, 81, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 100, 101, 102, 104,105, 107, 109, 111, 113, 115, 117, 118, 120, 122, 124, 126, 128, 130,132, 133, 135, 136, 138, 139, 141, 143, 145, 147, 148, 149, 151, 152,153, 155, 157, 158, 160, 161, 162, 163, 164, 165, 167, 168, 170, 171,172, 173, 175, 177, 179, 181, 182, 184, 185, 187, 189, 190, 192, 194,196, 197, 199, 201, 202, 204, 205, 207, 208, 210, 211, 213, 215, 216,218, 220, 221, 222, 223, 225, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 251, 252, 254, 256, 258, 260, 261, 262, 264,266, 268, 270, 272, 274, 275, 276, 278, 280, 282, 283, 284, 285, 286,288, 289, 290, 292, 294, 295, 296, 297, 298, 299, 300, 302, 303, 305,306, 308, 309, 310, 311, 312, 314, 316, 317, 318, 320, 321, 323, 325,326, 327, 328, 329, 331, 333, 335, 336, 337, 338, 339, 340, 342, 344,346, 348, 350, 351, 353, 355, 357, 359, 360, 361, 362, 363, 364, 365,366, 368, 370, 372, 373, 374, 375, 376, 377, 379, 380, 381, 383, 384,386, 388, 390, 392, 393, 394, 396, 398, 400, 402, 404, 406, 407, 408,410, 412, 413, 414, 416, 418, 419, 420, 422, 423, 424, 426, 427, 429,430, 431, 433, 434, 436, 437, 439, 441, 442, 444, 446, 448, 449, 450,451, 452, 454, 456, 458, 459, 460, 462, 463, 465, 466, 468, 470, 472,473, 474, 476, 478, 479, 480, 481, 483, 485, 486, 488, 490, 492, 493,495, 497, 498, 500, 501, 503, 504, 506, 508, 509, 510, 512, 514, 515,516, 517, 519, 521, 522, 523, 525, 527, 529, 531, 533, 534, 535, 537,539, 541, 543, 545, 547, 549, 551, 552, 554, 556, 557, 559, 562, 564,565, 567, 568, 570, 572, 573, 574, 575, 576, 578, 580, 582, 584, 586,588, 589, 590, 592, 594, 596, 598, 599, 601, 602, 603, 605, 607, 608,609, 611, 613, 615, 617, 619, 621, 622, 624, 625, 627, 629, 630, 632,634, 636, 638, 641, 643, 645, 646, 647, 648, 649, 650, 651, 652, 653,654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 677, 679,680, 682, 684, 686, 687, 688, 689, 690, 691, 692, 694, 695, 697, 699,701, 702, 704, 706, 708, 710, 712, 713, 715, 716, 718, 720, 721, 723,724, 726, 727, 729, 730, 732, 733, 735, 736, 737, 739, 740, 742, 744,746, 747, 748, 749, 750, 751, 753, 755, 757, 758, 760, 761, 763, 764,765, 766, 767, 768, 769, 771, 772, 774, 775, 776, 777, 778, 780, 781,782, 783, 785, 786, 788, 789, 791, 793, 795, 796, 798, 799, 800, 801,802, 803, 805, 807, 808, 810, 811, 812, 813, 814, 815, 816, 817, 818,819, 820, 821, and 823. A plant produced from the plant cell has adifference in biomass composition as compared to the correspondingcomposition of a control plant that does not include the nucleic acid.The difference in biomass composition in the plant can be a differencein the sucrose content or conversion efficiency.

In the methods described herein, the polypeptide can include aheavy-metal-associated domain having 60 percent or greater sequenceidentity to residues 6 to 73 of SEQ ID NO: 562. The polypeptide caninclude a Myb-like DNA-binding domain having 60 percent or greatersequence identity to residues 212 to 263 of SEQ ID NO: 246. Thepolypeptide can include a DUF1070 domain having 60 percent or greatersequence identity to residues 4-52 of SEQ ID NO: 111. The polypeptidecan include a glycosyl hydrolase family 16 domain and a xyloglucanendo-transglycosylase (XET) domain having 60 percent or greater sequenceidentity to residues 39 to 224 and 246 to 292 of SEQ ID NO: 348,respectively. The polypeptide can include an Alpha-L-AF_C domain having60 percent or greater sequence identity to residues 454 to 643 of SEQ IDNO: 774 and a CBM_4_9 domain having 60 percent or greater sequenceidentity to residues 71 to 229 of SEQ ID NO: 774. The polypeptide caninclude a COBRA domain having 60 percent or greater sequence identity toresidues 45 to 209 of SEQ ID NO: 416. The polypeptide can include aglycosyl transferase family 8 domain having 60 percent or greatersequence identity to residues 30 to 253 of SEQ ID NO: 2. The polypeptidecan include a DUF563 domain having 60 percent or greater sequenceidentity to residues 196 to 439 of SEQ ID NO: 157. The polypeptide caninclude an XG_FTase domain having 60 percent or greater sequenceidentity to residues 72 to 574 of SEQ ID NO: 280. The polypeptide caninclude a glycosyl hydrolase family 16 domain having 60 percent orgreater sequence identity to residues 23 to 204 of SEQ ID NO: 641 and aXET domain having 60 percent or greater sequence identity to residues228 to 280 of SEQ ID NO: 641. The polypeptide can include a potatoinhibitor I family domain having 60 percent or greater sequence identityto residues 17 to 76 of SEQ ID NO: 26.

In the methods described herein, the polypeptide can be selected fromthe group consisting of SEQ ID NOs: 2, 4, 6, 7, 8, 10, 12, 14, 15, 17,18, 19, 20, 21, 22, 24, 26, 28, 29, 30, 32, 34, 36, 37, 39, 41, 43, 45,47, 49, 50, 52, 53, 55, 57, 59, 61, 63, 65, 66, 68, 70, 71, 72, 73, 75,77, 78, 79, 81, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 100, 101, 102,104, 105, 107, 109, 111, 113, 115, 117, 118, 120, 122, 124, 126, 128,130, 132, 133, 135, 136, 138, 139, 141, 143, 145, 147, 148, 149, 151,152, 153, 155, 157, 158, 160, 161, 162, 163, 164, 165, 167, 168, 170,171, 172, 173, 175, 177, 179, 181, 182, 184, 185, 187, 189, 190, 192,194, 196, 197, 199, 201, 202, 204, 205, 207, 208, 210, 211, 213, 215,216, 218, 220, 221, 222, 223, 225, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 251, 252, 254, 256, 258, 260, 261, 262,264, 266, 268, 270, 272, 274, 275, 276, 278, 280, 282, 283, 284, 285,286, 288, 289, 290, 292, 294, 295, 296, 297, 298, 299, 300, 302, 303,305, 306, 308, 309, 310, 311, 312, 314, 316, 317, 318, 320, 321, 323,325, 326, 327, 328, 329, 331, 333, 335, 336, 337, 338, 339, 340, 342,344, 346, 348, 350, 351, 353, 355, 357, 359, 360, 361, 362, 363, 364,365, 366, 368, 370, 372, 373, 374, 375, 376, 377, 379, 380, 381, 383,384, 386, 388, 390, 392, 393, 394, 396, 398, 400, 402, 404, 406, 407,408, 410, 412, 413, 414, 416, 418, 419, 420, 422, 423, 424, 426, 427,429, 430, 431, 433, 434, 436, 437, 439, 441, 442, 444, 446, 448, 449,450, 451, 452, 454, 456, 458, 459, 460, 462, 463, 465, 466, 468, 470,472, 473, 474, 476, 478, 479, 480, 481, 483, 485, 486, 488, 490, 492,493, 495, 497, 498, 500, 501, 503, 504, 506, 508, 509, 510, 512, 514,515, 516, 517, 519, 521, 522, 523, 525, 527, 529, 531, 533, 534, 535,537, 539, 541, 543, 545, 547, 549, 551, 552, 554, 556, 557, 559, 562,564, 565, 567, 568, 570, 572, 573, 574, 575, 576, 578, 580, 582, 584,586, 588, 589, 590, 592, 594, 596, 598, 599, 601, 602, 603, 605, 607,608, 609, 611, 613, 615, 617, 619, 621, 622, 624, 625, 627, 629, 630,632, 634, 636, 638, 641, 643, 645, 646, 647, 648, 649, 650, 651, 652,653, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 677,679, 680, 682, 684, 686, 687, 688, 689, 690, 691, 692, 694, 695, 697,699, 701, 702, 704, 706, 708, 710, 712, 713, 715, 716, 718, 720, 721,723, 724, 726, 727, 729, 730, 732, 733, 735, 736, 737, 739, 740, 742,744, 746, 747, 748, 749, 750, 751, 753, 755, 757, 758, 760, 761, 763,764, 765, 766, 767, 768, 769, 771, 772, 774, 775, 776, 777, 778, 780,781, 782, 783, 785, 786, 788, 789, 791, 793, 795, 796, 798, 799, 800,801, 802, 803, 805, 807, 808, 810, 811, 812, 813, 814, 815, 816, 817,818, 819, 820, 821, and 823.

This document also features a method of modulating the biomasscomposition in a plant. The method includes introducing into a plantcell an exogenous nucleic acid, the exogenous nucleic acid comprising aregulatory region operably linked to a nucleotide sequence having 80percent or greater sequence identity to a nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 1, 3, 5, 9, 11, 13, 16, 23, 25,27, 31, 33, 35, 38, 40, 42, 44, 46, 48, 51, 54, 56, 58, 60, 62, 64, 67,69, 74, 76, 80, 83, 85, 87, 89, 91, 93, 95, 98, 103, 106, 108, 110, 112,114, 116, 119, 121, 123, 125, 127, 129, 131, 134, 137, 140, 142, 144,146, 150, 154, 156, 159, 166, 169, 174, 176, 178, 180, 183, 186, 188,191, 193, 195, 198, 200, 203, 206, 209, 212, 214, 217, 219, 224, 227,229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 253, 255, 257,259, 263, 265, 267, 269, 271, 273, 277, 279, 281, 287, 291, 293, 301,304, 307, 313, 315, 319, 322, 324, 330, 332, 334, 341, 343, 345, 347,349, 352, 354, 356, 358, 367, 369, 371, 378, 382, 385, 387, 389, 391,395, 397, 399, 401, 403, 405, 409, 411, 415, 417, 421, 425, 428, 432,435, 438, 440, 443, 445, 447, 453, 455, 457, 461, 464, 467, 469, 471,475, 477, 482, 484, 487, 489, 491, 494, 496, 499, 502, 505, 507, 511,513, 518, 520, 524, 526, 528, 530, 532, 536, 538, 540, 542, 544, 546,548, 550, 553, 555, 558, 560, 561, 563, 566, 569, 571, 577, 579, 581,583, 585, 587, 591, 593, 595, 597, 600, 604, 606, 610, 612, 614, 616,618, 620, 623, 626, 628, 631, 633, 635, 637, 639, 640, 642, 644, 655,657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 678, 681, 683, 685,693, 696, 698, 700, 703, 705, 707, 709, 711, 714, 717, 719, 722, 725,728, 731, 734, 738, 741, 743, 745, 752, 754, 756, 759, 762, 770, 773,779, 784, 787, 790, 792, 794, 797, 804, 806, 809, and 822, or a fragmentthereof A plant produced from the plant cell has a difference in biomasscomposition as compared to the corresponding composition of a controlplant that does not comprise the nucleic acid. The difference in biomasscomposition in the plant can be a difference in the sucrose content orconversion efficiency.

In another aspect, this document features a plant cell that includes anexogenous nucleic acid. The exogenous nucleic acid includes a regulatoryregion operably linked to a nucleotide sequence encoding a polypeptide,wherein the HMM bit score of the amino acid sequence of the polypeptideis greater than about 65, where the HMM is based on the amino acidsequences depicted in one of FIGS. 1-12 , and wherein a plant producedfrom the plant cell has a difference in biomass composition as comparedto the corresponding composition of a control plant that does notcomprise the nucleic acid. The difference in biomass composition in theplant can be a difference in the sucrose content or conversionefficiency.

This document also features a plant cell that includes an exogenousnucleic acid, where the exogenous nucleic acid includes a regulatoryregion operably linked to a nucleotide sequence encoding a polypeptidehaving 80 percent or greater sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 4, 6, 7, 8, 10, 12,14, 15, 17, 18, 19, 20, 21, 22, 24, 26, 28, 29, 30, 32, 34, 36, 37, 39,41, 43, 45, 47, 49, 50, 52, 53, 55, 57, 59, 61, 63, 65, 66, 68, 70, 71,72, 73, 75, 77, 78, 79, 81, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 100,101, 102, 104, 105, 107, 109, 111, 113, 115, 117, 118, 120, 122, 124,126, 128, 130, 132, 133, 135, 136, 138, 139, 141, 143, 145, 147, 148,149, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 164, 165, 167,168, 170, 171, 172, 173, 175, 177, 179, 181, 182, 184, 185, 187, 189,190, 192, 194, 196, 197, 199, 201, 202, 204, 205, 207, 208, 210, 211,213, 215, 216, 218, 220, 221, 222, 223, 225, 226, 228, 230, 232, 234,236, 238, 240, 242, 244, 246, 248, 250, 251, 252, 254, 256, 258, 260,261, 262, 264, 266, 268, 270, 272, 274, 275, 276, 278, 280, 282, 283,284, 285, 286, 288, 289, 290, 292, 294, 295, 296, 297, 298, 299, 300,302, 303, 305, 306, 308, 309, 310, 311, 312, 314, 316, 317, 318, 320,321, 323, 325, 326, 327, 328, 329, 331, 333, 335, 336, 337, 338, 339,340, 342, 344, 346, 348, 350, 351, 353, 355, 357, 359, 360, 361, 362,363, 364, 365, 366, 368, 370, 372, 373, 374, 375, 376, 377, 379, 380,381, 383, 384, 386, 388, 390, 392, 393, 394, 396, 398, 400, 402, 404,406, 407, 408, 410, 412, 413, 414, 416, 418, 419, 420, 422, 423, 424,426, 427, 429, 430, 431, 433, 434, 436, 437, 439, 441, 442, 444, 446,448, 449, 450, 451, 452, 454, 456, 458, 459, 460, 462, 463, 465, 466,468, 470, 472, 473, 474, 476, 478, 479, 480, 481, 483, 485, 486, 488,490, 492, 493, 495, 497, 498, 500, 501, 503, 504, 506, 508, 509, 510,512, 514, 515, 516, 517, 519, 521, 522, 523, 525, 527, 529, 531, 533,534, 535, 537, 539, 541, 543, 545, 547, 549, 551, 552, 554, 556, 557,559, 562, 564, 565, 567, 568, 570, 572, 573, 574, 575, 576, 578, 580,582, 584, 586, 588, 589, 590, 592, 594, 596, 598, 599, 601, 602, 603,605, 607, 608, 609, 611, 613, 615, 617, 619, 621, 622, 624, 625, 627,629, 630, 632, 634, 636, 638, 641, 643, 645, 646, 647, 648, 649, 650,651, 652, 653, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674,676, 677, 679, 680, 682, 684, 686, 687, 688, 689, 690, 691, 692, 694,695, 697, 699, 701, 702, 704, 706, 708, 710, 712, 713, 715, 716, 718,720, 721, 723, 724, 726, 727, 729, 730, 732, 733, 735, 736, 737, 739,740, 742, 744, 746, 747, 748, 749, 750, 751, 753, 755, 757, 758, 760,761, 763, 764, 765, 766, 767, 768, 769, 771, 772, 774, 775, 776, 777,778, 780, 781, 782, 783, 785, 786, 788, 789, 791, 793, 795, 796, 798,799, 800, 801, 802, 803, 805, 807, 808, 810, 811, 812, 813, 814, 815,816, 817, 818, 819, 820, 821, and 823, wherein a plant produced from theplant cell has a difference in biomass composition as compared to thecorresponding composition of a control plant that does not comprise thenucleic acid. The difference in biomass composition in the plant can bea difference in the sucrose content or conversion efficiency.

In yet another aspect, this document features a plant cell that includesan exogenous nucleic acid. The exogenous nucleic acid includes aregulatory region operably linked to a nucleotide sequence having 80percent or greater sequence identity to a nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 1, 3, 5, 9, 11, 13, 16, 23, 25,27, 31, 33, 35, 38, 40, 42, 44, 46, 48, 51, 54, 56, 58, 60, 62, 64, 67,69, 74, 76, 80, 83, 85, 87, 89, 91, 93, 95, 98, 103, 106, 108, 110, 112,114, 116, 119, 121, 123, 125, 127, 129, 131, 134, 137, 140, 142, 144,146, 150, 154, 156, 159, 166, 169, 174, 176, 178, 180, 183, 186, 188,191, 193, 195, 198, 200, 203, 206, 209, 212, 214, 217, 219, 224, 227,229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 253, 255, 257,259, 263, 265, 267, 269, 271, 273, 277, 279, 281, 287, 291, 293, 301,304, 307, 313, 315, 319, 322, 324, 330, 332, 334, 341, 343, 345, 347,349, 352, 354, 356, 358, 367, 369, 371, 378, 382, 385, 387, 389, 391,395, 397, 399, 401, 403, 405, 409, 411, 415, 417, 421, 425, 428, 432,435, 438, 440, 443, 445, 447, 453, 455, 457, 461, 464, 467, 469, 471,475, 477, 482, 484, 487, 489, 491, 494, 496, 499, 502, 505, 507, 511,513, 518, 520, 524, 526, 528, 530, 532, 536, 538, 540, 542, 544, 546,548, 550, 553, 555, 558, 560, 561, 563, 566, 569, 571, 577, 579, 581,583, 585, 587, 591, 593, 595, 597, 600, 604, 606, 610, 612, 614, 616,618, 620, 623, 626, 628, 631, 633, 635, 637, 639, 640, 642, 644, 655,657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 678, 681, 683, 685,693, 696, 698, 700, 703, 705, 707, 709, 711, 714, 717, 719, 722, 725,728, 731, 734, 738, 741, 743, 745, 752, 754, 756, 759, 762, 770, 773,779, 784, 787, 790, 792, 794, 797, 804, 806, 809, and 822, or a fragmentthereof, wherein a plant produced from the plant cell has a differencein biomass composition as compared to the corresponding composition of acontrol plant that does not comprise the nucleic acid. The difference inbiomass composition in the plant can be a difference in the sucrosecontent or conversion efficiency.

This document also features a transgenic plant comprising any of theplant cells described herein. The plant can be a member of a speciesselected from the group consisting of Panicum virgatum (switchgrass),Sorghum bicolor (sorghum, sudangrass), Miscanthus giganteus(miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar),Zea mays (corn), Glycine max (soybean), Brassica napus (canola),Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa(rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Betavulgaris (sugarbeet), and Pennisetum glaucum (pearl millet). Atransgenic plant can include a polypeptide selected from the groupconsisting of SEQ ID NO: 2, 4, 6, 7, 8, 10, 12, 14, 15, 17, 18, 19, 20,21, 22, 24, 26, 28, 29, 30, 32, 34, 36, 37, 39, 41, 43, 45, 47, 49, 50,52, 53, 55, 57, 59, 61, 63, 65, 66, 68, 70, 71, 72, 73, 75, 77, 78, 79,81, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 100, 101, 102, 104, 105,107, 109, 111, 113, 115, 117, 118, 120, 122, 124, 126, 128, 130, 132,133, 135, 136, 138, 139, 141, 143, 145, 147, 148, 149, 151, 152, 153,155, 157, 158, 160, 161, 162, 163, 164, 165, 167, 168, 170, 171, 172,173, 175, 177, 179, 181, 182, 184, 185, 187, 189, 190, 192, 194, 196,197, 199, 201, 202, 204, 205, 207, 208, 210, 211, 213, 215, 216, 218,220, 221, 222, 223, 225, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 248, 250, 251, 252, 254, 256, 258, 260, 261, 262, 264, 266,268, 270, 272, 274, 275, 276, 278, 280, 282, 283, 284, 285, 286, 288,289, 290, 292, 294, 295, 296, 297, 298, 299, 300, 302, 303, 305, 306,308, 309, 310, 311, 312, 314, 316, 317, 318, 320, 321, 323, 325, 326,327, 328, 329, 331, 333, 335, 336, 337, 338, 339, 340, 342, 344, 346,348, 350, 351, 353, 355, 357, 359, 360, 361, 362, 363, 364, 365, 366,368, 370, 372, 373, 374, 375, 376, 377, 379, 380, 381, 383, 384, 386,388, 390, 392, 393, 394, 396, 398, 400, 402, 404, 406, 407, 408, 410,412, 413, 414, 416, 418, 419, 420, 422, 423, 424, 426, 427, 429, 430,431, 433, 434, 436, 437, 439, 441, 442, 444, 446, 448, 449, 450, 451,452, 454, 456, 458, 459, 460, 462, 463, 465, 466, 468, 470, 472, 473,474, 476, 478, 479, 480, 481, 483, 485, 486, 488, 490, 492, 493, 495,497, 498, 500, 501, 503, 504, 506, 508, 509, 510, 512, 514, 515, 516,517, 519, 521, 522, 523, 525, 527, 529, 531, 533, 534, 535, 537, 539,541, 543, 545, 547, 549, 551, 552, 554, 556, 557, 559, 562, 564, 565,567, 568, 570, 572, 573, 574, 575, 576, 578, 580, 582, 584, 586, 588,589, 590, 592, 594, 596, 598, 599, 601, 602, 603, 605, 607, 608, 609,611, 613, 615, 617, 619, 621, 622, 624, 625, 627, 629, 630, 632, 634,636, 638, 641, 643, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654,656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 677, 679, 680,682, 684, 686, 687, 688, 689, 690, 691, 692, 694, 695, 697, 699, 701,702, 704, 706, 708, 710, 712, 713, 715, 716, 718, 720, 721, 723, 724,726, 727, 729, 730, 732, 733, 735, 736, 737, 739, 740, 742, 744, 746,747, 748, 749, 750, 751, 753, 755, 757, 758, 760, 761, 763, 764, 765,766, 767, 768, 769, 771, 772, 774, 775, 776, 777, 778, 780, 781, 782,783, 785, 786, 788, 789, 791, 793, 795, 796, 798, 799, 800, 801, 802,803, 805, 807, 808, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819,820, 821, and 823. A seed product can include embryonic tissue from atransgenic plant described herein.

This document also features an isolated nucleic acid that includes anucleotide sequence having 85% or greater sequence identity to thenucleotide sequence set forth in SEQ ID NO: 9, 13, 16, 23, 166, 169,186, 198, 212, 219, 229, 231, 235, 265, 267, 269, 287, 307, 313, 322,324, 330, 332, 334, 341, 343, 354, 356, 385, 387, 389, 395, 401, 411,542, 550, 553, 558, 571, 579, 585, 591, 593, 597, 600, 606, 614, 618,623, 628, 631, 635, or 637.

In another aspect, an isolated nucleic acid is featured that includes anucleotide sequence encoding a polypeptide having 80% or greatersequence identity to the amino acid sequence set forth in SEQ ID NO: 8,10, 14, 15, 17, 21, 22, 24, 57, 167, 170, 187, 213, 220, 230, 232, 236,266, 268, 270, 285, 286, 288, 290, 295, 296, 297, 299, 308, 309, 310,311, 314, 317, 318, 323, 325, 327, 329, 331, 333, 335, 338, 342, 344,355, 357, 360, 362, 363, 364, 366, 374, 377, 381, 386, 388, 390, 392,393, 394, 396, 402, 408, 412, 413, 414, 493, 543, 551, 554, 557, 559,572, 573, 574, 575, 586, 589, 590, 592, 594, 598, 599, 601, 602, 603,607, 609, 615, 619, 622, 624, 625, 629, 630, 632, 636, 638, 776, 814,815, 816, 817, 818, 819, 820, or 821.

This document also features a method of identifying whether apolymorphism is associated with variation in a trait. The methodincludes determining whether one or more genetic polymorphisms in apopulation of plants is associated with the locus for a polypeptideselected from the group consisting of the polypeptides depicted in FIGS.1-12 and functional homologs thereof; and measuring the correlationbetween variation in the trait in plants of the population and thepresence of one or more genetic polymorphisms in plants of thepopulation, thereby identifying whether or not the one or more geneticpolymorphisms are associated with variation in the trait. The variationin biomass composition can be a variation in sucrose content orconversion efficiency. The population can be a population of switchgrassplants.

In another aspect, this document features a method of making a plantline. The method includes determining whether one or more geneticpolymorphisms in a population of plants is associated with the locus fora polypeptide selected from the group consisting of the polypeptidesdepicted in FIGS. 1-12 and functional homologs thereof; identifying oneor more plants in the population in which the presence of at least oneof the genetic polymorphisms is associated with variation in biomasscomposition; crossing one or more of the identified plants with itselfor a different plant to produce seed; crossing at least one progenyplant grown from the seed with itself or a different plant; andrepeating the crossing steps for an additional 0-5 generations to makethe plant line, wherein at least one of the genetic polymorphisms ispresent in the plant line. The variation in biomass composition can be avariation in sucrose content or conversion efficiency. The populationcan be a population of switchgrass plants.

This document also features a method of altering biomass composition ina plant. The method includes modifying an endogenous biomasscomposition-modulating nucleic acid, the nucleic acid comprising anucleotide sequence with an open reading frame having 80 percent orgreater (e.g., 90 percent or greater, or 95 percent or greater) sequenceidentity to the nucleotide sequence selected from the group consistingof SEQ ID NO: 1, 3, 5, 9, 11, 13, 16, 23, 25, 27, 31, 33, 35, 38, 40,42, 44, 46, 48, 51, 54, 56, 58, 60, 62, 64, 67, 69, 74, 76, 80, 83, 85,87, 89, 91, 93, 95, 98, 103, 106, 108, 110, 112, 114, 116, 119, 121,123, 125, 127, 129, 131, 134, 137, 140, 142, 144, 146, 150, 154, 156,159, 166, 169, 174, 176, 178, 180, 183, 186, 188, 191, 193, 195, 198,200, 203, 206, 209, 212, 214, 217, 219, 224, 227, 229, 231, 233, 235,237, 239, 241, 243, 245, 247, 249, 253, 255, 257, 259, 263, 265, 267,269, 271, 273, 277, 279, 281, 287, 291, 293, 301, 304, 307, 313, 315,319, 322, 324, 330, 332, 334, 341, 343, 345, 347, 349, 352, 354, 356,358, 367, 369, 371, 378, 382, 385, 387, 389, 391, 395, 397, 399, 401,403, 405, 409, 411, 415, 417, 421, 425, 428, 432, 435, 438, 440, 443,445, 447, 453, 455, 457, 461, 464, 467, 469, 471, 475, 477, 482, 484,487, 489, 491, 494, 496, 499, 502, 505, 507, 511, 513, 518, 520, 524,526, 528, 530, 532, 536, 538, 540, 542, 544, 546, 548, 550, 553, 555,558, 560, 561, 563, 566, 569, 571, 577, 579, 581, 583, 585, 587, 591,593, 595, 597, 600, 604, 606, 610, 612, 614, 616, 618, 620, 623, 626,628, 631, 633, 635, 637, 639, 640, 642, 644, 655, 657, 659, 661, 663,665, 667, 669, 671, 673, 675, 678, 681, 683, 685, 693, 696, 698, 700,703, 705, 707, 709, 711, 714, 717, 719, 722, 725, 728, 731, 734, 738,741, 743, 745, 752, 754, 756, 759, 762, 770, 773, 779, 784, 787, 790,792, 794, 797, 804, 806, 809, and 822, wherein the plant has adifference in biomass composition as compared to the correspondingcomposition of a control plant where the nucleic acid has not beenmodified. The modification can be effected by introducing a geneticmodification in the locus comprising the nucleic acid. The methodfurther can include selecting for plants having altered biomasscomposition. The endogenous nucleic acid can encode a polypeptide having80 percent or greater (e.g., 90 percent or greater, or 95 percent orgreater) sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, 4, 6, 7, 8, 10, 12, 14, 15, 17, 18,19, 20, 21, 22, 24, 26, 28, 29, 30, 32, 34, 36, 37, 39, 41, 43, 45, 47,49, 50, 52, 53, 55, 57, 59, 61, 63, 65, 66, 68, 70, 71, 72, 73, 75, 77,78, 79, 81, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 100, 101, 102, 104,105, 107, 109, 111, 113, 115, 117, 118, 120, 122, 124, 126, 128, 130,132, 133, 135, 136, 138, 139, 141, 143, 145, 147, 148, 149, 151, 152,153, 155, 157, 158, 160, 161, 162, 163, 164, 165, 167, 168, 170, 171,172, 173, 175, 177, 179, 181, 182, 184, 185, 187, 189, 190, 192, 194,196, 197, 199, 201, 202, 204, 205, 207, 208, 210, 211, 213, 215, 216,218, 220, 221, 222, 223, 225, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 251, 252, 254, 256, 258, 260, 261, 262, 264,266, 268, 270, 272, 274, 275, 276, 278, 280, 282, 283, 284, 285, 286,288, 289, 290, 292, 294, 295, 296, 297, 298, 299, 300, 302, 303, 305,306, 308, 309, 310, 311, 312, 314, 316, 317, 318, 320, 321, 323, 325,326, 327, 328, 329, 331, 333, 335, 336, 337, 338, 339, 340, 342, 344,346, 348, 350, 351, 353, 355, 357, 359, 360, 361, 362, 363, 364, 365,366, 368, 370, 372, 373, 374, 375, 376, 377, 379, 380, 381, 383, 384,386, 388, 390, 392, 393, 394, 396, 398, 400, 402, 404, 406, 407, 408,410, 412, 413, 414, 416, 418, 419, 420, 422, 423, 424, 426, 427, 429,430, 431, 433, 434, 436, 437, 439, 441, 442, 444, 446, 448, 449, 450,451, 452, 454, 456, 458, 459, 460, 462, 463, 465, 466, 468, 470, 472,473, 474, 476, 478, 479, 480, 481, 483, 485, 486, 488, 490, 492, 493,495, 497, 498, 500, 501, 503, 504, 506, 508, 509, 510, 512, 514, 515,516, 517, 519, 521, 522, 523, 525, 527, 529, 531, 533, 534, 535, 537,539, 541, 543, 545, 547, 549, 551, 552, 554, 556, 557, 559, 562, 564,565, 567, 568, 570, 572, 573, 574, 575, 576, 578, 580, 582, 584, 586,588, 589, 590, 592, 594, 596, 598, 599, 601, 602, 603, 605, 607, 608,609, 611, 613, 615, 617, 619, 621, 622, 624, 625, 627, 629, 630, 632,634, 636, 638, 641, 643, 645, 646, 647, 648, 649, 650, 651, 652, 653,654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 677, 679,680, 682, 684, 686, 687, 688, 689, 690, 691, 692, 694, 695, 697, 699,701, 702, 704, 706, 708, 710, 712, 713, 715, 716, 718, 720, 721, 723,724, 726, 727, 729, 730, 732, 733, 735, 736, 737, 739, 740, 742, 744,746, 747, 748, 749, 750, 751, 753, 755, 757, 758, 760, 761, 763, 764,765, 766, 767, 768, 769, 771, 772, 774, 775, 776, 777, 778, 780, 781,782, 783, 785, 786, 788, 789, 791, 793, 795, 796, 798, 799, 800, 801,802, 803, 805, 807, 808, 810, 811, 812, 813, 814, 815, 816, 817, 818,819, 820, 821, and 823.

This document also features a method of producing a plant. The methodincludes growing a plant cell containing a modified endogenous nucleicacid encoding a polypeptide, wherein the HMM bit score of the amino acidsequence of the polypeptide is greater than about 65, the HMM based onthe amino acid sequences depicted in one of FIGS. 1-12 , and wherein theplant has a difference in biomass composition as compared to thecorresponding composition of a control plant where the nucleic acid hasnot been modified.

In another aspect, this document features a plant cell containing amodified endogenous nucleic acid encoding a polypeptide, wherein the HMMbit score of the amino acid sequence of the polypeptide is greater thanabout 65, the HMM based on the amino acid sequences depicted in one ofFIGS. 1-12 , and wherein a plant produced from the plant cell has adifference in biomass composition as compared to the correspondingcomposition of a control plant where the nucleic acid has not beenmodified.

In yet another aspect, this document features a plant cell containing amodified biomass composition-modulating endogenous nucleic acid. Thenucleic acid includes a nucleotide sequence with an open reading framehaving 80 percent or greater sequence identity to the nucleotidesequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 9,11, 13, 16, 23, 25, 27, 31, 33, 35, 38, 40, 42, 44, 46, 48, 51, 54, 56,58, 60, 62, 64, 67, 69, 74, 76, 80, 83, 85, 87, 89, 91, 93, 95, 98, 103,106, 108, 110, 112, 114, 116, 119, 121, 123, 125, 127, 129, 131, 134,137, 140, 142, 144, 146, 150, 154, 156, 159, 166, 169, 174, 176, 178,180, 183, 186, 188, 191, 193, 195, 198, 200, 203, 206, 209, 212, 214,217, 219, 224, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 253, 255, 257, 259, 263, 265, 267, 269, 271, 273, 277, 279, 281,287, 291, 293, 301, 304, 307, 313, 315, 319, 322, 324, 330, 332, 334,341, 343, 345, 347, 349, 352, 354, 356, 358, 367, 369, 371, 378, 382,385, 387, 389, 391, 395, 397, 399, 401, 403, 405, 409, 411, 415, 417,421, 425, 428, 432, 435, 438, 440, 443, 445, 447, 453, 455, 457, 461,464, 467, 469, 471, 475, 477, 482, 484, 487, 489, 491, 494, 496, 499,502, 505, 507, 511, 513, 518, 520, 524, 526, 528, 530, 532, 536, 538,540, 542, 544, 546, 548, 550, 553, 555, 558, 560, 561, 563, 566, 569,571, 577, 579, 581, 583, 585, 587, 591, 593, 595, 597, 600, 604, 606,610, 612, 614, 616, 618, 620, 623, 626, 628, 631, 633, 635, 637, 639,640, 642, 644, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675,678, 681, 683, 685, 693, 696, 698, 700, 703, 705, 707, 709, 711, 714,717, 719, 722, 725, 728, 731, 734, 738, 741, 743, 745, 752, 754, 756,759, 762, 770, 773, 779, 784, 787, 790, 792, 794, 797, 804, 806, 809,and 822, and wherein a plant produced from the plant cell has adifference in biomass composition as compared to the correspondingcomposition of a control plant where the nucleic acid has not beenmodified. The difference in biomass composition in the plant can be adifference in the sucrose content or conversion efficiency.

An endogenous nucleic acid can encode a polypeptide having 80 percent orgreater sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, 4, 6, 7, 8, 10, 12, 14, 15, 17, 18,19, 20, 21, 22, 24, 26, 28, 29, 30, 32, 34, 36, 37, 39, 41, 43, 45, 47,49, 50, 52, 53, 55, 57, 59, 61, 63, 65, 66, 68, 70, 71, 72, 73, 75, 77,78, 79, 81, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 100, 101, 102, 104,105, 107, 109, 111, 113, 115, 117, 118, 120, 122, 124, 126, 128, 130,132, 133, 135, 136, 138, 139, 141, 143, 145, 147, 148, 149, 151, 152,153, 155, 157, 158, 160, 161, 162, 163, 164, 165, 167, 168, 170, 171,172, 173, 175, 177, 179, 181, 182, 184, 185, 187, 189, 190, 192, 194,196, 197, 199, 201, 202, 204, 205, 207, 208, 210, 211, 213, 215, 216,218, 220, 221, 222, 223, 225, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 251, 252, 254, 256, 258, 260, 261, 262, 264,266, 268, 270, 272, 274, 275, 276, 278, 280, 282, 283, 284, 285, 286,288, 289, 290, 292, 294, 295, 296, 297, 298, 299, 300, 302, 303, 305,306, 308, 309, 310, 311, 312, 314, 316, 317, 318, 320, 321, 323, 325,326, 327, 328, 329, 331, 333, 335, 336, 337, 338, 339, 340, 342, 344,346, 348, 350, 351, 353, 355, 357, 359, 360, 361, 362, 363, 364, 365,366, 368, 370, 372, 373, 374, 375, 376, 377, 379, 380, 381, 383, 384,386, 388, 390, 392, 393, 394, 396, 398, 400, 402, 404, 406, 407, 408,410, 412, 413, 414, 416, 418, 419, 420, 422, 423, 424, 426, 427, 429,430, 431, 433, 434, 436, 437, 439, 441, 442, 444, 446, 448, 449, 450,451, 452, 454, 456, 458, 459, 460, 462, 463, 465, 466, 468, 470, 472,473, 474, 476, 478, 479, 480, 481, 483, 485, 486, 488, 490, 492, 493,495, 497, 498, 500, 501, 503, 504, 506, 508, 509, 510, 512, 514, 515,516, 517, 519, 521, 522, 523, 525, 527, 529, 531, 533, 534, 535, 537,539, 541, 543, 545, 547, 549, 551, 552, 554, 556, 557, 559, 562, 564,565, 567, 568, 570, 572, 573, 574, 575, 576, 578, 580, 582, 584, 586,588, 589, 590, 592, 594, 596, 598, 599, 601, 602, 603, 605, 607, 608,609, 611, 613, 615, 617, 619, 621, 622, 624, 625, 627, 629, 630, 632,634, 636, 638, 641, 643, 645, 646, 647, 648, 649, 650, 651, 652, 653,654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 677, 679,680, 682, 684, 686, 687, 688, 689, 690, 691, 692, 694, 695, 697, 699,701, 702, 704, 706, 708, 710, 712, 713, 715, 716, 718, 720, 721, 723,724, 726, 727, 729, 730, 732, 733, 735, 736, 737, 739, 740, 742, 744,746, 747, 748, 749, 750, 751, 753, 755, 757, 758, 760, 761, 763, 764,765, 766, 767, 768, 769, 771, 772, 774, 775, 776, 777, 778, 780, 781,782, 783, 785, 786, 788, 789, 791, 793, 795, 796, 798, 799, 800, 801,802, 803, 805, 807, 808, 810, 811, 812, 813, 814, 815, 816, 817, 818,819, 820, 821, and 823, and wherein a plant produced from the plant cellhas a difference in biomass composition as compared to the correspondingcomposition of a control plant where the nucleic acid has not beenmodified. The difference in biomass composition in the plant can be adifference in the sucrose content or conversion efficiency.

This document also features a plant cell that includes an exogenousnucleic acid, the exogenous nucleic acid encoding a polypeptide havingE.C. 3.2.1.55 activity, and wherein a plant produced from the plant cellhas a difference in biomass composition as compared to the correspondinglevel of a control plant that does not comprise said nucleic acid. Thedifference in biomass composition in the plant can be a difference inthe sucrose content or conversion efficiency.

In another aspect, this document features a method of modulating biomasscomposition of a plant. The method includes introducing into a plantcell an exogenous nucleic acid, the exogenous nucleic acid encoding apolypeptide having E.C. 3.2.1.55 activity.

This document also features a process for making a biofuel. The processincludes planting seeds of a sorghum plant described herein, or asorghum plant produced by a method described herein in one or morefields to obtain at least about 10 acres of the sorghum plants;harvesting sorghum biomass from the one or more fields to obtainharvested sorghum biomass; extracting sorghum stem juice from theharvested sorghum biomass to obtain extracted stem juice comprisingsugar; using said sugar of the extracted stem juice in a fermentationreaction to produce a fermentation product comprising a biofuel; andisolating the biofuel from the fermentation product to obtain acomposition comprising the biofuel (e.g., ethanol or anhydrous ethanol).The sorghum plants can have an average BRIX value that is greater thanabout 10 percent at harvest time.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims. The word “comprising” inthe claims may be replaced by “consisting essentially of” or with“consisting of,” according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1 is an alignment of the amino acid sequence of CeresClone: 1767521(SEQ ID NO: 483) with homologous and/or orthologous amino acidsequences. In all the alignment figures shown herein, a dash in analigned sequence represents a gap, i.e., a lack of an amino acid at thatposition. Identical amino acids or conserved amino acid substitutionsamong aligned sequences are identified by boxes. FIG. 1 and the otheralignment figures provided herein were generated using the programMUSCLE version 3.52.

FIGS. 2A-2C are an alignment of the amino acid sequence of CeresClone:1871180 (SEQ ID NO: 562) with homologous and/or orthologous amino acidsequences.

FIGS. 3A-3C are an alignment of the amino acid sequence of CeresClone:240112 (SEQ ID NO: 246) with homologous and/or orthologous amino acidsequences.

FIG. 4 are an alignment of the amino acid sequence of CeresClone:1764605 (SEQ ID NO:111) with homologous and/or orthologous amino acidsequences.

FIGS. 5A-5E are an alignment of the amino acid sequence of CeresClone:1776501 (SEQ ID NO: 348) with homologous and/or orthologous amino acidsequences.

FIGS. 6A-6I are an alignment of the amino acid sequence of CeresClone:1789981 (SEQ ID NO: 774) with homologous and/or orthologous amino acidsequences.

FIGS. 7A-7G are an alignment of the amino acid sequence of CeresClone:1804732 (SEQ ID NO: 416) with homologous and/or orthologous amino acidsequences.

FIGS. 8A-8E are an alignment of the amino acid sequence of CeresClone:1807011 (SEQ ID NO: 2) with homologous and/or orthologous amino acidsequences.

FIGS. 9A-9I are an alignment of the amino acid sequence of CeresClone:1888614 (SEQ ID NO: 157) with homologous and/or orthologous amino acidsequences.

FIGS. 10A-10G are an alignment of the amino acid sequence of CeresClone:1900192 (SEQ ID NO:280) with homologous and/or orthologous amino acidsequences.

FIGS. 11A-11D are an alignment of the amino acid sequence of CeresClone:1955550 (SEQ ID NO: 641) with homologous and/or orthologous amino acidsequences.

FIG. 12 is an alignment of the amino acid sequence of CeresClone:1955766(SEQ ID NO: 26) with homologous and/or orthologous amino acid sequences.

DETAILED DESCRIPTION

This document features methods and materials related to modulatingbiomass composition (e.g., sucrose content or conversion efficiency) inplants. For example, this document features methods and materials forincreasing or decreasing sucrose content and conversion efficiency inplants. In some embodiments, the plants also may have modulated levelsof, for example, lignin, modified root architecture, modified herbicideresistance, or modified carotenoid biosynthesis. The methods can includetransforming a plant cell with a nucleic acid encoding a biomasscomposition-modulating polypeptide, wherein expression of thepolypeptide results in modulated biomass composition. Plant cellsproduced using such methods can be grown to produce plants having anincreased or decreased sucrose content and/or conversion efficiency.Such plants may produce more grazable forage. Increased brix levels (anapproximate amount of sugar as measured by, for example, a digitalrefractometer) and/or sucrose content can result in increasedpalatability as a forage crop. In addition, such plants, and the seedsof such plants, may be used to produce, for example, switchgrass,miscanthus, Sorghum sp., and sugar cane plants having increased value asa biofuel feedstock.

I. DEFINITIONS

“Accessible Carbohydrate” refers to mono- and oligo-saccharides releasedinto the aqueous phase after processing of a biomass feedstock. Theamount of accessible carbohydrate in a feedstock is related to thepretreatment and enzymatic saccharification conditions chosen for thesaccharification process and to the composition and structure of theinitial biomass feedstock.

“Amino acid” refers to one of the twenty biologically occurring aminoacids and to synthetic amino acids, including D/L optical isomers.

“Ash” refers to inorganic material that contributes to the dry weight ofthe feedstock. Ash content in biomass feedstocks can be determined usingpublished, standard methods such as ASTM Standard E1755.

“Biofuels” include, but are not limited to, biodiesel, methanol,ethanol, butanol, linear alkanes (C₅-C₂₀), branched-chain alkanes(C₅-C₂₆), mixed alkanes, linear alcohols (C₁-C₂₀), branched-chainalcohols (C₁-C₂₆), linear carboxylic acids (C₂-C₂₀), and branched-chaincarboxylic acids (C₂-C₂₆). In addition, ethers, esters and amides of theaforementioned acids and alcohols, as well as other conjugates of thesechemicals may be of interest. Many of these chemicals can besubsequently converted by chemical reactions to other high value, highvolume chemicals.

“Biomass” refers to organic matter. Biomass includes plant matterderived from herbaceous and woody energy crops, agricultural food andfeed crops, agricultural crop wastes and residues, wood wastes andresidues, aquatic plants, and other plant-derived materials. Biomass mayalso include algae, yard wastes, and include some municipal wastes.Biomass is a heterogeneous and chemically complex renewable resource.Components of biomass include glucan, xylan, fermentable sugars,arabinan, sucrose, lignin, protein, ash, extractives, ferulate, andacetate.

“Cell type-preferential promoter” or “tissue-preferential promoter”refers to a promoter that drives expression preferentially in a targetcell type or tissue, respectively, but may also lead to sometranscription in other cell types or tissues as well.

“Control plant” refers to a plant that does not contain the exogenousnucleic acid present in a transgenic plant of interest, but otherwisehas the same or similar genetic background as such a transgenic plant. Asuitable control plant can be a non-transgenic wild type plant, anon-transgenic segregant from a transformation experiment, or atransgenic plant that contains an exogenous nucleic acid other than theexogenous nucleic acid of interest.

“Conversion efficiency” refers to the conversion of biomass feedstock tofree sugars, fermentable sugars, syngas, biofuel, ethanol, heat, orenergy in a laboratory-, pilot-, or production-scale process. Therelevant conversion efficiency parameters are dependent on the type ofconversion process employed (biochemical, thermochemical to biofuel, orthermochemical to heat and electricity). NIR spectra of biomass samplesare collected and translated by a NIR model (see below) to predictfeedstock conversion properties (such as free sugars or accessiblecarbohydrate), one or more intermediate values that may serve forpredicting feedstock conversion properties (such as recalcitrantcarbohydrate), or one or more downstream parameters that are influencedby feedstock conversion efficiency (such as biofuel or energy yield).Predictions of conversion properties may be used to calculate thefeedstock performance characteristics in one or more processing methodsof interest. Such performance characteristics include saccharificationefficiency or sugar yield (Glu, Xyl, Ara, Man, Gal), various enzymaticconditions (type, ratio, load) for saccharification, pretreatmentconditions, total or net energy yield or energy conversion efficiency,biofuel yield or biofuel conversion efficiency, biopower yield orbiopower conversion efficiency, coproduct yield or extraction/conversionefficiency, economic value of the original feedstock, NOX emissions,protein coproducts, or sustainability indicators.

“Domains” are groups of substantially contiguous amino acids in apolypeptide that can be used to characterize protein families and/orparts of proteins. Such domains have a “fingerprint” or “signature” thatcan comprise conserved primary sequence, secondary structure, and/orthree-dimensional conformation. Generally, domains are correlated withspecific in vitro and/or in vivo activities. A domain can have a lengthof from 10 amino acids to 400 amino acids, e.g., 10 to 50 amino acids,or 25 to 100 amino acids, or 35 to 65 amino acids, or 35 to 55 aminoacids, or 45 to 60 amino acids, or 200 to 300 amino acids, or 300 to 400amino acids.

“Down-regulation” refers to regulation that decreases production ofexpression products (mRNA, polypeptide, or both) relative to basal ornative states.

“Exogenous” with respect to a nucleic acid indicates that the nucleicacid is part of a recombinant nucleic acid construct, or is not in itsnatural environment. For example, an exogenous nucleic acid can be asequence from one species introduced into another species, i.e., aheterologous nucleic acid. Typically, such an exogenous nucleic acid isintroduced into the other species via a recombinant nucleic acidconstruct. An exogenous nucleic acid can also be a sequence that isnative to an organism and that has been reintroduced into cells of thatorganism. An exogenous nucleic acid that includes a native sequence canoften be distinguished from the naturally occurring sequence by thepresence of non-natural sequences linked to the exogenous nucleic acid,e.g., non-native regulatory sequences flanking a native sequence in arecombinant nucleic acid construct. In addition, stably transformedexogenous nucleic acids typically are integrated at positions other thanthe position where the native sequence is found. It will be appreciatedthat an exogenous nucleic acid may have been introduced into aprogenitor and not into the cell under consideration. For example, atransgenic plant containing an exogenous nucleic acid can be the progenyof a cross between a stably transformed plant and a non-transgenicplant. Such progeny are considered to contain the exogenous nucleicacid.

“Expression” refers to the process of converting genetic information ofa polynucleotide into RNA through transcription, which is catalyzed byan enzyme, RNA polymerase, and into protein, through translation of mRNAon ribosomes.

“Glucan,” “Xylan” and “Arabinan” refer to the anhydro forms of glucose,xylose and arabinose that are found in cellulose and hemicellulosecarbohydrate polymers. Thus, for example, “glucan” refers to apolysaccharide of D-glucose monomers linked by glycosidic bonds. Thefollowing are glucans: cellulose (β-1,4-glucan), dextran (α-1,6-glucan)and starch (α-1,4- and α-1,6-glucan).

“Hemicellulose” is a general term used to refer to cell wallpolysaccharides that are not celluloses or pectins. Hemicellulosescontain repeating monomeric units of a five-carbon sugar (usuallyD-xylose or L-arabinose) and/or a six-carbon sugar (D-galactose,D-glucose, and D-mannose). See, U.S. Pat. No. 7,112,429. Hemicellulosestypically are shorter in length than cellulose and are highly branched.Xylan is often the structural backbone of hemicelluloses from hardwoodsand grasses, and hydrolysis of these biomass types releases productshigh in the five-carbon sugar, xylose. Hemicelluloses from softwoods aremost commonly gluco-galacto-mannans, which have a mannan backbone andyield mannose as the main product of hydrolysis. Hemicelluloses oftencontain side groups such as acetyl groups, uronic acids and ferulates.

“Heterologous polypeptide” as used herein refers to a polypeptide thatis not a naturally occurring polypeptide in a plant cell, e.g., atransgenic Panicum virgatum plant transformed with and expressing thecoding sequence for a nitrogen transporter polypeptide from a Zea maysplant.

“Higher heating value” (HHV) refers to the amount of heat released by aspecified quantity of a fuel at an initial temperature of 25° C.,following combustion, and return of the combustion products to atemperature of 25° C. The HHV is also known as the gross calorific valueor gross energy.

“Isolated nucleic acid” as used herein includes a naturally-occurringnucleic acid, provided one or both of the sequences immediately flankingthat nucleic acid in its naturally-occurring genome is removed orabsent. Thus, an isolated nucleic acid includes, without limitation, anucleic acid that exists as a purified molecule or a nucleic acidmolecule that is incorporated into a vector or a virus. A nucleic acidexisting among hundreds to millions of other nucleic acids within, forexample, cDNA libraries, genomic libraries, or gel slices containing agenomic DNA restriction digest, is not to be considered an isolatednucleic acid.

“Lignin” refers to a polyphenolic polymeric substance of plant cells,with a complex, cross-linked, highly aromatic structure. Lignin issynthesized in plants principally from three monolignol monomers, whichcan be methoxylated to various degrees: sinapyl alcohol (C₁₁H₁₄O₄) thatis incorporated into lignin as (S) syringyl units; coniferyl alcohol(C₁₀H₁₂O₃) that is incorporated into lignin as (G) guaiacyl units; andp-coumaryl alcohol (C₉H₁₀O₂) that is incorporated into lignin as (H)p-hydroxyphenyl units. These monomers can be synthesized into lignin byextensive condensation polymerization. The lignin present in differentplant varieties can have different syringyl:guaiacyl:p-hydroxyphenylweight percents (S:G:H weight percents). For example, certain grassvarieties can have lignin composed almost entirely of guaiacyl (G).Lignin is a major structural constituent of plant cells in woodyspecies.

“Modulation” of the level of biomass refers to the change in the levelof the biomass that is observed as a result of expression of, ortranscription from, an exogenous nucleic acid in a plant cell and/orplant. The change in level is measured relative to the correspondinglevel in control plants.

“NIR Model” refers to a series of validated mathematical equations thatpredict the chemical composition of a sample, based on NIR spectral datafrom the sample. The term also refers to a series of validatedmathematical equations that predict saccharification conversionefficiency of a sample, based on NIR spectral data from the sample. Inthe case of saccharification conversion efficiency, a different NIRmodel is developed for each combination of pretreatment conditions andenzyme(s). NIR spectral data typically is obtained from the sample at aplurality of different wavelengths, and the mathematical equations areapplied to the spectral data to calculate the predicted value. Thecalibration equations can be derived by regression among spectroscopicdata for feedstock samples of the same type, e.g., by multiple-linearregression, by partial least squares, or by neural network analysis.

“NOX emissions” refers to mono-nitrogen oxides (NOx), such as NO andNO2, released into the atmosphere. While oxygen and nitrogen gases donot typically react at ambient temperatures, oxygen and nitrogen gasescan react at higher temperatures to create various oxides of nitrogen,including mono-nitrogen oxides. Mono-nitrogen oxides can also beproduced by combusting materials including elemental nitrogen.Mono-nitrogen oxides (NOx) released into the atmosphere can react withvolatile organic compounds to produce smog. Accordingly, NOX emissionsmay be regulated by various governmental agencies. Oxides of sulfur(SOx), specifically sulfur dioxide, are often generated in the sameprocesses. SOx emissions are known to contribute to acid rain.

“Nucleic acid” and “polynucleotide” are used interchangeably herein, andrefer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA,and DNA or RNA containing nucleic acid analogs. A nucleic acid can bedouble-stranded or single-stranded (i.e., a sense strand or an antisensestrand). Non-limiting examples of polynucleotides include genes, genefragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomalRNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides,branched polynucleotides, nucleic acid probes and nucleic acid primers.A polynucleotide may contain unconventional or modified nucleotides.

“Operably linked” refers to the positioning of a regulatory region and asequence to be transcribed in a nucleic acid so that the regulatoryregion is effective for regulating transcription or translation of thesequence. For example, to operably link a coding sequence and aregulatory region, the translation initiation site of the translationalreading frame of the coding sequence is typically positioned between oneand about fifty nucleotides downstream of the regulatory region. Aregulatory region can, however, be positioned as much as about 5,000nucleotides upstream of the translation initiation site, or about 2,000nucleotides upstream of the transcription start site.

“Polypeptide” as used herein refers to a compound of two or more subunitamino acids, amino acid analogs, or other peptidomimetics, regardless ofpost-translational modification, e.g., phosphorylation or glycosylation.The subunits may be linked by peptide bonds or other bonds such as, forexample, ester or ether bonds. Full-length polypeptides, truncatedpolypeptides, point mutants, insertion mutants, splice variants,chimeric proteins, and fragments thereof are encompassed by thisdefinition.

“Progeny” includes descendants of a particular plant or plant line.Progeny of an instant plant include seeds formed on F₁, F₂, F₃, F₄, F₅,F₆ and subsequent generation plants, or seeds formed on BC₁, BC₂, BC₃,and subsequent generation plants, or seeds formed on F₁BC₁, F₁BC₂,F₁BC₃, and subsequent generation plants. The designation F₁ refers tothe progeny of a cross between two parents that are geneticallydistinct. The designations F₂, F₃, F₄, F₅ and F₆ refer to subsequentgenerations of self- or sib-pollinated progeny of an F₁ plant.

“Recalcitrant carbohydrate material” refers to mono- andoligo-saccharides that are not released into the aqueous phase afterprocessing of a biomass feedstock. It is related to the pretreatment andenzymatic saccharification conditions chosen for the saccharificationprocess.

“Regulatory region” refers to a nucleic acid having nucleotide sequencesthat influence transcription or translation initiation and rate, andstability and/or mobility of a transcription or translation product.Regulatory regions include, without limitation, promoter sequences,enhancer sequences, response elements, protein recognition sites,inducible elements, protein binding sequences, 5′ and 3′ untranslatedregions (UTRs), transcriptional start sites, termination sequences,polyadenylation sequences, introns, and combinations thereof Aregulatory region typically comprises at least a core (basal) promoter.A regulatory region also may include at least one control element, suchas an enhancer sequence, an upstream element or an upstream activationregion (UAR). For example, a suitable enhancer is a cis-regulatoryelement (−212 to −154) from the upstream region of the octopine synthase(ocs) gene. Fromm et al., The Plant Cell, 1:977-984 (1989).

“Saccharification” refers to the hydrolysis of carbohydrate material tothe mono- and disaccharides that constitute the polymer. For example,saccharification of xylan results in the production of xylose, themonosaccharide constituent of xylan. Saccharification occurs during thebiochemical processing of biomass in biorefineries, ultimately leadingto the production of biofuels such as ethanol.

“Saccharification efficiency” of a feedstock sample refers to the totalamount of mono and disaccharides solubilized by pretreatment/enzymaticsaccharification processes, divided by the theoretical maximum amount ofmono and disaccharides in the biomass sample that could have beenreleased based on compositional analysis, converted to a percentage bymultiplying by 100.

“Sustainability indicators” refer to components of biomass processingbyproducts, such as the expected ash composition and soil nutrients,which may be recycled.

“Up-regulation” refers to regulation that increases the level of anexpression product (mRNA, polypeptide, or both) relative to basal ornative states.

“Vector” refers to a replicon, such as a plasmid, phage, or cosmid, intowhich another DNA segment may be inserted so as to bring about thereplication of the inserted segment. Generally, a vector is capable ofreplication when associated with the proper control elements. The term“vector” includes cloning and expression vectors, as well as viralvectors and integrating vectors. An “expression vector” is a vector thatincludes a regulatory region.

II. POLYPEPTIDES

Polypeptides described herein include biomass composition-modulatingpolypeptides. Biomass composition-modulating polypeptides can beeffective to modulate biomass composition when expressed in a plant orplant cell. Such polypeptides typically contain at least one domainindicative of a biomass composition-modulating polypeptide, as describedin more detail herein. Biomass composition-modulating polypeptides alsotypically have an HMM bit score that is greater than 65 as described inmore detail herein. In some embodiments, biomass composition-modulatingpolypeptides have greater than 80% identity to SEQ ID NOs: 2, 4, 6, 7,8, 10, 12, 14, 15, 17, 18, 19, 20, 21, 22, 24, 26, 28, 29, 30, 32, 34,36, 37, 39, 41, 43, 45, 47, 49, 50, 52, 53, 55, 57, 59, 61, 63, 65, 66,68, 70, 71, 72, 73, 75, 77, 78, 79, 81, 82, 84, 86, 88, 90, 92, 94, 96,97, 99, 100, 101, 102, 104, 105, 107, 109, 111, 113, 115, 117, 118, 120,122, 124, 126, 128, 130, 132, 133, 135, 136, 138, 139, 141, 143, 145,147, 148, 149, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 164,165, 167, 168, 170, 171, 172, 173, 175, 177, 179, 181, 182, 184, 185,187, 189, 190, 192, 194, 196, 197, 199, 201, 202, 204, 205, 207, 208,210, 211, 213, 215, 216, 218, 220, 221, 222, 223, 225, 226, 228, 230,232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 251, 252, 254, 256,258, 260, 261, 262, 264, 266, 268, 270, 272, 274, 275, 276, 278, 280,282, 283, 284, 285, 286, 288, 289, 290, 292, 294, 295, 296, 297, 298,299, 300, 302, 303, 305, 306, 308, 309, 310, 311, 312, 314, 316, 317,318, 320, 321, 323, 325, 326, 327, 328, 329, 331, 333, 335, 336, 337,338, 339, 340, 342, 344, 346, 348, 350, 351, 353, 355, 357, 359, 360,361, 362, 363, 364, 365, 366, 368, 370, 372, 373, 374, 375, 376, 377,379, 380, 381, 383, 384, 386, 388, 390, 392, 393, 394, 396, 398, 400,402, 404, 406, 407, 408, 410, 412, 413, 414, 416, 418, 419, 420, 422,423, 424, 426, 427, 429, 430, 431, 433, 434, 436, 437, 439, 441, 442,444, 446, 448, 449, 450, 451, 452, 454, 456, 458, 459, 460, 462, 463,465, 466, 468, 470, 472, 473, 474, 476, 478, 479, 480, 481, 483, 485,486, 488, 490, 492, 493, 495, 497, 498, 500, 501, 503, 504, 506, 508,509, 510, 512, 514, 515, 516, 517, 519, 521, 522, 523, 525, 527, 529,531, 533, 534, 535, 537, 539, 541, 543, 545, 547, 549, 551, 552, 554,556, 557, 559, 562, 564, 565, 567, 568, 570, 572, 573, 574, 575, 576,578, 580, 582, 584, 586, 588, 589, 590, 592, 594, 596, 598, 599, 601,602, 603, 605, 607, 608, 609, 611, 613, 615, 617, 619, 621, 622, 624,625, 627, 629, 630, 632, 634, 636, 638, 641, 643, 645, 646, 647, 648,649, 650, 651, 652, 653, 654, 656, 658, 660, 662, 664, 666, 668, 670,672, 674, 676, 677, 679, 680, 682, 684, 686, 687, 688, 689, 690, 691,692, 694, 695, 697, 699, 701, 702, 704, 706, 708, 710, 712, 713, 715,716, 718, 720, 721, 723, 724, 726, 727, 729, 730, 732, 733, 735, 736,737, 739, 740, 742, 744, 746, 747, 748, 749, 750, 751, 753, 755, 757,758, 760, 761, 763, 764, 765, 766, 767, 768, 769, 771, 772, 774, 775,776, 777, 778, 780, 781, 782, 783, 785, 786, 788, 789, 791, 793, 795,796, 798, 799, 800, 801, 802, 803, 805, 807, 808, 810, 811, 812, 813,814, 815, 816, 817, 818, 819, 820, 821, and 823 as described in moredetail herein.

A. Domains Indicative of Biomass Composition-Modulating Polypeptides

A biomass composition-modulating polypeptide can contain amethyltransferase_2 domain and a dimerization domain, which arepredicted to be characteristic of a biomass composition-modulatingpolypeptide. SEQ ID NO: 562 sets forth the amino acid sequence of aPanicum virgatum clone, identified herein as CeresClone:1871180 (SEQ IDNO:561), that is predicted to encode a polypeptide containing aheavy-metal-associated domain. For example, a biomasscomposition-modulating polypeptide can comprise a heavy-metal-associateddomain having 60 percent or greater (e.g., 65, 70, 75, 80, 85, 90, 95,97, 98, 99, or 100 percent) sequence identity to residues 6 to 73 of SEQID NO: 562. In some embodiments, a biomass composition-modulatingpolypeptide can comprise a heavy-metal-associated domain having 60percent or greater (e.g., 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100percent) sequence identity to the heavy-metal-associated domain of oneor more of the polypeptides set forth in SEQ ID NOs: 564, 565, 567, 568,570, 572, 573, 574, 575, 576, 578, 580, 582, 584, 586, 588, 589, 590,592, 594, 596, 598, 599, 601, 602, 603, 605, 607, 608, 609, 611, 613,615, 617, 619, 621, 622, 624, 625, 627, 629, 630, 632, 634, 636, and638. The heavy-metal-associated domains of such sequences are set forthin the Sequence Listing. The heavy-metal-associated domain ischaracteristic of proteins that transport heavy metals, and typicallycontains two conserved cysteines that may be involved in metal binding.See, e.g., Rosenzweig et al., Structure Fold Des., 7:605-617 (1999).

A biomass composition-modulating polypeptide can contain a Myb-likeDNA-binding domain, which is predicted to be characteristic of a biomasscomposition-modulating polypeptide. A polypeptide containing such aMyb-like DNA-binding domain can be useful, for example, for modulatingsucrose content or conversion efficiency. SEQ ID NO: 246 sets forth theamino acid sequence of a Zea mays clone, identified herein asCeresClone:240112 (SEQ ID NO: 245) that is predicted to encode apolypeptide containing a Myb-like DNA-binding domain. For example, abiomass composition-modulating polypeptide can comprise a Myb-likeDNA-binding domain having 60 percent or greater (e.g., 65, 70, 75, 80,85, 90, 95, 97, 98, 99, or 100 percent) sequence identity to residues212 to 263 of SEQ ID NO: 246. In some embodiments, a biomasscomposition-modulating polypeptide can comprise a Myb-like DNA-bindingdomain having 60 percent or greater (e.g., 65, 70, 75, 80, 85, 90, 95,97, 98, 99, or 100 percent) sequence identity to the Myb-likeDNA-binding domain of one or more of the polypeptides set forth in SEQID NOs: 248, 250, 251, 252, 254, 256, 258, 260, 261, 262, 264, 266, 268,270, 272, 274, 275, 276, and 278. The Myb-like DNA-binding domains ofsuch sequences are set forth in the Sequence Listing. TheMyb_DNA-binding domain is found in the family of Myb proteins, as wellas the SANT domain family. See, Aasland et al., Trends Biochem Sci121:87-88 (1996). The SANT domain family specifically recognizes thesequence YAAC(G/T)G.

A biomass composition-modulating polypeptide can contain a DUF1070domain, which is predicted to be characteristic of a biomasscomposition-modulating polypeptide. A polypeptide containing such aDUF1070 domain can be useful, for example, for modulating sucrosecontent. SEQ ID NO: 111 sets forth the amino acid sequence of a Panicumvirgatum clone, identified herein as CeresClone:1764605 (SEQ ID NO:110)that is predicted to encode a polypeptide containing a DUF1070 domain.For example, a biomass composition-modulating polypeptide can comprise aDUF1070 domain having 60 percent or greater (e.g., 65, 70, 75, 80, 85,90, 95, 97, 98, 99, or 100 percent) sequence identity to residues 4-52of SEQ ID NO: 111. In some embodiments, a biomass composition-modulatingpolypeptide can comprise a DUF1070 domain having 60 percent or greater(e.g., 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100 percent) sequenceidentity to the DUF1070 domain of one or more of the polypeptides setforth in SEQ ID NOs: 113, 115, 117, 118, 120, 122, 124, 126, 128, 130,132, 133, 135, 136, 138, 139, 141, 143, 145, 147, 148, 149, 151, 152,153, 155. The DUF1070 domain is a conserved domain found in severalshort plant proteins, including the arabinogalactan peptide family. See,e.g., Schultz et al., Plant Cell 12:1751-68 (2000).

A biomass composition-modulating polypeptide can contain a glycosylhydrolases family 16 domain and a xyloglucan endo-transglycosylase (XET)domain, which are predicted to be characteristic of a biomasscomposition-modulating polypeptide. A polypeptide containing such aglycosyl hydrolases family 16 domain and XET domain can be useful, forexample, for modulating sucrose content or conversion efficiency. SEQ IDNO: 348 sets forth the amino acid sequence of a Panicum virgatum clone,identified herein as CeresClone:1776501 (SEQ ID NO: 347), that ispredicted to encode a polypeptide containing a glycosyl hydrolasesfamily 16 domain and a XET domain. For example, a biomasscomposition-modulating polypeptide can comprise a glycosyl hydrolasesfamily 16 domain and a XET domain having 60 percent or greater (e.g.,65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100 percent) sequenceidentity to residues 39 to 224 and 246 to 292 of SEQ ID NO: 348,respectively. In some embodiments, a biomass composition-modulatingpolypeptide can comprise a glycosyl hydrolases family 16 domain and aXET domain having 60 percent or greater (e.g., 65, 70, 75, 80, 85, 90,95, 97, 98, 99, or 100 percent) sequence identity to the glycosylhydrolases family 16 domain and XET domain of one or more of thepolypeptides set forth in SEQ ID NOs: 350, 351, 353, 355, 357, 359, 360,361, 362, 363, 364, 365, 366, 368, 370, 372, 373, 374, 375, 376, 377,379, 380, 381, 383, 384, 386, 388, 390, 392, 393, 394, 396, 398, 400,402, 404, 406, 407, 408, 410, 412, 413, and 414. The glycosyl hydrolasesfamily 16 domain and XET domain of such sequences are set forth in theSequence Listing. Proteins within the glycosyl hydrolase family 16 areO-glycosyl hydrolases that hydrolyze the glycosidic bond between two ormore carbohydrates, or between a carbohydrate and a non-carbohydratemoiety. Members of the glycosyl hydrolase 16 family include lichenase,xyloglucan xyloglucosyltransferase, agarase, kappa-carrageenase,endo-beta-1,3-glucanase, endo-beta-1,3-1,4-glucanase, andendo-beta-galactosidase. The XET domain is found in the C-terminus(approximately 60 residues) of plant xyloglucan endo-transglycosylases.Xyloglucan is the predominant hemicellulose in the cell walls of mostdicotyledons. With cellulose, it forms a network that strengthens thecell wall. XET catalyzes the splitting of xyloglucan chains and thelinking of the newly generated reducing end to the non-reducing end ofanother xyloglucan chain, thereby loosening the cell wall. See, forexample, Schroder et al., Planta, 204:242-251 (1998).

A biomass composition-modulating polypeptide can contain an Alpha-L-AF_Cdomain and a CBM_4_9 domain, which are predicted to be characteristic ofa biomass composition-modulating polypeptide. A polypeptide containingsuch an Alpha-L-AF_C domain and a CBM_4_9 domain can be useful, forexample, for modulating sucrose content or conversion efficiency. SEQ IDNO: 774 sets forth the amino acid sequence of a Panicum virgatum clone,identified herein as CeresClone:1789981 (SEQ ID NO: 773), that ispredicted to encode a polypeptide containing Alpha-L-AF_C and CBM_4_9domains. For example, a biomass composition-modulating polypeptide cancomprise an Alpha-L-AF_C domain having 60 percent or greater (e.g., 65,70, 75, 80, 85, 90, 95, 97, 98, 99, or 100 percent) sequence identity toresidues 454 to 643 of SEQ ID NO: 774 and a CBM_4_9 domain having 60percent or greater (e.g., 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100percent) sequence identity to residues 71 to 229 of SEQ ID NO: 774. Insome embodiments, a biomass composition-modulating polypeptide cancomprise Alpha-L-AF_C and CBM_4_9 domains having 60 percent or greater(e.g., 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100 percent) sequenceidentity to the Alpha-L-AF_C and CBM_4_9 domains of one or more of thepolypeptides set forth in SEQ ID NOs: 775, 776, 777, 778, 780, 781, 782,783, 785, 786, 788, 789, 791, 793, 795, 796, 798, 799, 800, 801, 802,803, 805, 807, 808, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819,820, and 821. The Alpha-L-AF_C and CBM_4_9 domains of such sequences areset forth in the Sequence Listing. The Alpha-L-AF_C domain representsthe approximately 200 C-terminal residues of bacterial and eukaryoticalpha-L-arabinofuranosidase (EC:3.2.1.55), which catalyzes thehydrolysis of nonreducing terminal alpha-L-arabinofuranosidic linkagesin L-arabinose-containing polysaccharides. The CBM_4_9 domain is acarbohydrate binding domain.

A biomass composition-modulating polypeptide can contain a COBRA domain,which is predicted to be characteristic of a biomasscomposition-modulating polypeptide. A polypeptide containing such aCOBRA domain can be useful, for example, for modulating sucrose contentor conversion efficiency. SEQ ID NO: 416 sets forth the amino acidsequence of a Panicum virgatum clone, identified herein asCeresClone:1804732 (SEQ ID NO: 415), that is predicted to encode apolypeptide containing a COBRA domain. For example, a biomasscomposition-modulating polypeptide can comprise a COBRA domain having 60percent or greater (e.g., 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100percent) sequence identity to residues 45 to 209 of SEQ ID NO: 416. Insome embodiments, a biomass composition-modulating polypeptide cancomprise a COBRA domain having 60 percent or greater (e.g., 65, 70, 75,80, 85, 90, 95, 97, 98, 99, or 100 percent) sequence identity to theCOBRA domain of one or more of the polypeptides set forth in SEQ ID NOs:418, 419, 420, 422, 423, 424, 426, 427, 429, 430, 431, 433, 434, 436,437, 439, 441, 442, 444, 446, 448, 449, 450, 451, 452, 454, 456, 458,459, 460, 462, 463, 465, 466, 468, 470, 472, 473, 474, 476, 478, 479,480, and 481. COBRA domains are found within a family of plant proteinsdesignated COBRA-like (COBL) proteins. Members of the family areextracellular glycosyl-phosphatidyl inositol-anchored proteins(GPI-linked). COBRA is involved in determining the orientation of cellexpansion, probably by playing an important role in cellulosedeposition. It may act by recruiting cellulose synthesizing complexes todiscrete positions on the cell surface. See Roudier et al., Plant Cell.17(6):1749-63 (2005), Epub 2005 Apr. 22.

A biomass composition-modulating polypeptide can contain a glycosyltransferase family 8 domain, which is predicted to be characteristic ofa biomass composition-modulating polypeptide. A polypeptide containingsuch a glycosyl transferase family 8 domain can be useful, for example,for modulating sucrose content. SEQ ID NO: 2 sets forth the amino acidsequence of a Panicum virgatum clone, identified herein asCeresClone:1807011 (SEQ ID NO: 1), that is predicted to encode apolypeptide containing a glycosyl transferase family 8 domain. Forexample, a biomass composition-modulating polypeptide can comprise aglycosyl transferase family 8 domain having 60 percent or greater (e.g.,65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100 percent) sequenceidentity to residues 30 to 253 of SEQ ID NO: 2. In some embodiments, abiomass composition-modulating polypeptide can comprise a glycosyltransferase family 8 domain having 60 percent or greater (e.g., 65, 70,75, 80, 85, 90, 95, 97, 98, 99, or 100 percent) sequence identity to theglycosyl transferase family 8 domain of one or more of the polypeptidesset forth in SEQ ID NOs: 4, 6, 7, 8, 10, 12, 14, 15, 17, 18, 19, 20, 21,22, and 24. The glycosyl transferase family 8 domains of such sequencesare set forth in the Sequence Listing. The glycosyl transferase family 8domain is found in a family of enzymes that transfer sugar residues todonor molecules. Members of this family include lipopolysaccharidegalactosyltransferase, lipopolysaccharide glucosyltransferase 1,glycogenin glucosyltransferase, and inositol1-alpha-galactosyltransferase. In some embodiments, a nucleic acidsequence encoding the amino acid sequence set forth in SEQ ID NO:2 or ahomolog thereof can include a mutation (e.g., a deletion of anucleotide) such that a truncated polypeptide is produced. For example,the nucleic acid sequence can include a mutation such that the aminoacid sequence set forth in SEQ ID NO:2 is truncated at about position142.

A biomass composition-modulating polypeptide can contain a DUF563domain, which is predicted to be characteristic of a biomasscomposition-modulating polypeptide. A polypeptide containing such aDUF563 domain can be useful, for example, for modulating sucrosecontent. SEQ ID NO: 157 sets forth the amino acid sequence of a Panicumvirgatum clone, identified herein as CeresClone:1888614 (SEQ ID NO:156), that is predicted to encode a polypeptide containing a DUF563domain. For example, a biomass composition-modulating polypeptide cancomprise a DUF563 domain having 60 percent or greater (e.g., 65, 70, 75,80, 85, 90, 95, 97, 98, 99, or 100 percent) sequence identity toresidues 196 to 439 of SEQ ID NO: 157. In some embodiments, a biomasscomposition-modulating polypeptide can comprise a DUF563 domain having60 percent or greater (e.g., 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or100 percent) sequence identity to the DUF563 domain of one or more ofthe polypeptides set forth in SEQ ID NOs: 158, 160, 161, 162, 163, 164,165, 167, 168, 170, 171, 172, 173, 175, 177, 179, 181, 182, 184, 185,187, 189, 190, 192, 194, 196, 197, 199, 201, 202, 204, 205, 207, 208,210, 211, 213, 215, 216, 218, 220, 221, 222, 223, 225, 226, 228, 230,232, 234, 236, 238, 240, 242, and 244. The DUF563 domains of suchsequences are set forth in the Sequence Listing. Proteins having aDUF563 domain are in glycosyltransferase family 61.

A biomass composition-modulating polypeptide can contain a xyloglucanfucosyltransferase (XG_FTase) domain, which is predicted to becharacteristic of a biomass composition-modulating polypeptide. Apolypeptide containing such an XG_FTase domain can be useful, forexample, for modulating sucrose content. SEQ ID NO: 280 sets forth theamino acid sequence of a Panicum virgatum clone, identified herein asCeresClone:1900192 (SEQ ID NO: 279), that is predicted to encode apolypeptide containing an XG_FTase domain. For example, a biomasscomposition-modulating polypeptide can comprise an XG_FTase domainhaving 60 percent or greater (e.g., 65, 70, 75, 80, 85, 90, 95, 97, 98,99, or 100 percent) sequence identity to residues 72 to 574 of SEQ IDNO: 280. In some embodiments, a biomass composition-modulatingpolypeptide can comprise an XG_FTase domain having 60 percent or greater(e.g., 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100 percent) sequenceidentity to the XG_FTase domain of one or more of the polypeptides setforth in SEQ ID NOs: 280, 282, 283, 284, 285, 286, 288, 289, 290, 292,294, 295, 296, 297, 298, 299, 300, 302, 303, 305, 306, 308, 309, 310,311, 312, 314, 316, 317, 318, 320, 321, 323, 325, 326, 327, 328, 329,331, 333, 335, 336, 337, 338, 339, 340, 342, 344, and 346. The XG_FTasedomains of such sequences are set forth in the Sequence Listing. TheXG_FTase domain is found in a fucosyltransferase transfers the terminalfucosyl residue to xyloglucan (XG), the principal load-bearinghemicellulose of dicotyledonous plants. See, e.g., Perrin et al.,Science, 284:1976-1979 (1999).

A biomass composition-modulating polypeptide can contain a glycosylhydrolase family 16 domain and a xyloglucan endo-transglycosylase (XET)domain, which are predicted to be characteristic of a biomasscomposition-modulating polypeptide. A polypeptide containing such aglycosyl hydrolase family 16 domain and XET domain can be useful, forexample, for modulating sucrose content or conversion efficiency. SEQ IDNO: 641 sets forth the amino acid sequence of a Panicum virgatum clone,identified herein as CeresClone:1955550 (SEQ ID NO: 640), that ispredicted to encode a polypeptide containing a glycosyl hydrolase family16 domain and a XET domain. For example, a biomasscomposition-modulating polypeptide can comprise a glycosyl hydrolasesfamily 16 domain having 60 percent or greater (e.g., 65, 70, 75, 80, 85,90, 95, 97, 98, 99, or 100 percent) sequence identity to residues 23 to204 of SEQ ID NO: 641 and a XET domain having 60 percent or greater(e.g., 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100 percent) sequenceidentity to residues 228 to 280 of SEQ ID NO: 641. In some embodiments,a biomass composition-modulating polypeptide can comprise glycosylhydrolases family 16 and XET domains having 60 percent or greater (e.g.,65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100 percent) sequenceidentity to the glycosyl hydrolase family 16 and XET domains of one ormore of the polypeptides set forth in SEQ ID NOs: 643, 645, 646, 647,648, 649, 650, 651, 652, 653, 654, 656, 658, 660, 662, 664, 666, 668,670, 672, 674, 676, 677, 679, 680, 682, 684, 686, 687, 688, 689, 690,691, 692, 694, 695, 697, 699, 701, 702, 704, 706, 708, 710, 712, 713,715, 716, 718, 720, 721, 723, 724, 726, 727, 729, 730, 732, 733, 735,736, 737, 739, 740, 742, 744, 746, 747, 748, 749, 750, 751, 753, 755,757, 758, 760, 761, 763, 764, 765, 766, 767, 768, 769, 771, 772, or 823.The glycosyl hydrolases family 16 and XET domains of such sequences areset forth in the Sequence Listing. The glycosyl hydrolases family 16domain and XET domain are described above with reference to SEQ IDNO:348.

A biomass composition-modulating polypeptide can contain a potatoinhibitor I family domain, which is predicted to be characteristic of abiomass composition-modulating polypeptide. A polypeptide containingsuch a potato inhibitor I family domain can be useful, for example, formodulating sucrose content. SEQ ID NO: 26 sets forth the amino acidsequence of a Panicum virgatum clone, identified herein asCeresClone:1955766 (SEQ ID NO: 25), that is predicted to encode apolypeptide containing a potato inhibitor I family domain. For example,a biomass composition-modulating polypeptide can comprise a potatoinhibitor I family domain having 60 percent or greater (e.g., 65, 70,75, 80, 85, 90, 95, 97, 98, 99, or 100 percent) sequence identity toresidues 17 to 76 of SEQ ID NO: 26. In some embodiments, a biomasscomposition-modulating polypeptide can comprise a potato inhibitor Ifamily domain having 60 percent or greater (e.g., 65, 70, 75, 80, 85,90, 95, 97, 98, 99, or 100 percent) sequence identity to the potatoinhibitor I family domain of one or more of the polypeptides set forthin SEQ ID NOs: 28, 29, 30, 32, 34, 36, 37, 39, 41, 43, 45, 47, 49, 50,52, 53, 55, 57, 59, 61, 63, 65, 66, 68, 70, 71, 72, 73, 75, 77, 78, 79,81, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 100, 101, 102, 104, 105,107, and 109. The potato inhibitor I family domains of such sequencesare set forth in the Sequence Listing. Members of the potato inhibitor Ifamily are proteinase inhibitors that inhibit peptidases of the S1 andS8 families. See, for example, Rawlings et al., Biochem J. 15, 378(Pt3):705-16 (2004). Inhibitors in this family are small (60 to 90residues) and lack disulfide bonds. Typically, the inhibitor is awedge-shaped molecule, its pointed edge formed by the protease-bindingloop, which contains the scissile bond. The loop binds tightly to theprotease active site, with subsequent cleavage of the scissile bondcausing inhibition of the enzyme. See, Bode et al., EMBO J., 5(4):813-8(1986).

In some embodiments, a biomass composition-modulating polypeptide istruncated at the amino- or carboxy-terminal end of a naturally occurringpolypeptide. A truncated polypeptide may retain certain domains of thenaturally occurring polypeptide while lacking others. Thus, lengthvariants that are up to 5 amino acids shorter or longer typicallyexhibit the biomass composition-modulating activity of a truncatedpolypeptide. In some embodiments, a truncated polypeptide is a dominantnegative polypeptide. Expression in a plant of such a truncatedpolypeptide confers a difference in biomass composition of a plant ascompared to the corresponding level of a control plant that does notcomprise the truncation.

B. Functional Homologs Identified by Reciprocal BLAST

In some embodiments, one or more functional homologs of a referencebiomass composition-modulating polypeptide defined by one or more of thePfam descriptions indicated above are suitable for use as biomasscomposition-modulating polypeptides. A functional homolog is apolypeptide that has sequence similarity to a reference polypeptide, andthat carries out one or more of the biochemical or physiologicalfunction(s) of the reference polypeptide. A functional homolog and thereference polypeptide may be natural occurring polypeptides, and thesequence similarity may be due to convergent or divergent evolutionaryevents. As such, functional homologs are sometimes designated in theliterature as homologs, or orthologs, or paralogs. Variants of anaturally occurring functional homolog, such as polypeptides encoded bymutants of a wild type coding sequence, may themselves be functionalhomologs. Functional homologs can also be created via site-directedmutagenesis of the coding sequence for a biomass composition-modulatingpolypeptide, or by combining domains from the coding sequences fordifferent naturally-occurring biomass composition-modulatingpolypeptides (“domain swapping”). The term “functional homolog” issometimes applied to the nucleic acid that encodes a functionallyhomologous polypeptide.

Functional homologs can be identified by analysis of nucleotide andpolypeptide sequence alignments. For example, performing a query on adatabase of nucleotide or polypeptide sequences can identify homologs ofbiomass composition-modulating polypeptides. Sequence analysis caninvolve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of nonredundantdatabases using a biomass composition-modulating polypeptide amino acidsequence as the reference sequence. Amino acid sequence is, in someinstances, deduced from the nucleotide sequence. Those polypeptides inthe database that have greater than 40% sequence identity are candidatesfor further evaluation for suitability as a biomasscomposition-modulating polypeptide Amino acid sequence similarity allowsfor conservative amino acid substitutions, such as substitution of onehydrophobic residue for another or substitution of one polar residue foranother. If desired, manual inspection of such candidates can be carriedout in order to narrow the number of candidates to be further evaluated.Manual inspection can be performed by selecting those candidates thatappear to have domains present in biomass composition-modulatingpolypeptides, e.g., conserved functional domains.

Conserved regions can be identified by locating a region within theprimary amino acid sequence of a biomass composition-modulatingpolypeptide that is a repeated sequence, forms some secondary structure(e.g., helices and beta sheets), establishes positively or negativelycharged domains, or represents a protein motif or domain. See, e.g., thePfam web site describing consensus sequences for a variety of proteinmotifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/and pfam.janelia.org/. A description of the information included at thePfam database is described in Sonnhammer et al., Nucl. Acids Res.,26:320-322 (1998); Sonnhammer et al., Proteins, 28:405-420 (1997); andBateman et al., Nucl. Acids Res., 27:260-262 (1999). Conserved regionsalso can be determined by aligning sequences of the same or relatedpolypeptides from closely related species. Closely related speciespreferably are from the same family. In some embodiments, alignment ofsequences from two different species is adequate.

Typically, polypeptides that exhibit at least about 40% amino acidsequence identity are useful to identify conserved regions. Conservedregions of related polypeptides exhibit at least 45% amino acid sequenceidentity (e.g., at least 50%, at least 60%, at least 70%, at least 80%,or at least 90% amino acid sequence identity). In some embodiments, aconserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acidsequence identity.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 483 are provided in FIG. 1 and inthe Sequence Listing. Such functional homologs include, for example,CeresAnnot:8701398 (SEQ ID NO: 485), GI:21741986 (SEQ ID NO: 486),CeresClone:488555 (SEQ ID NO: 488), CeresAnnot:1472210 (SEQ ID NO: 490),CeresClone:1839543 (SEQ ID NO: 492), GI:124360895 (SEQ ID NO: 493),CeresClone:1778664 (SEQ ID NO: 495), CeresClone:2030878 (SEQ ID NO:497), GI:115458882 (SEQ ID NO: 498), CeresAnnot:8701404 (SEQ ID NO:500), GI:115458830 (SEQ ID NO: 501), CeresAnnot:8701387 (SEQ ID NO:503), GI:116310418 (SEQ ID NO: 504), CeresAnnot:8679943 (SEQ ID NO:506), CeresAnnot:8701391 (SEQ ID NO: 508), GI:46806257 (SEQ ID NO: 509),GI:125540058 (SEQ ID NO: 510), CeresClone:1018979 (SEQ ID NO: 512),CeresClone:1725423 (SEQ ID NO: 514), GI:115446965 (SEQ ID NO: 515),GI:125540059 (SEQ ID NO: 516), GI:38606531 (SEQ ID NO: 517),CeresClone:1955791 (SEQ ID NO: 519), CeresClone:2032166 (SEQ ID NO:521), GI:125540060 (SEQ ID NO: 522), GI:46806261 (SEQ ID NO: 523),CeresClone:100178733 (SEQ ID NO: 525), CeresClone:351547 (SEQ ID NO:527), CeresClone:1906874 (SEQ ID NO: 529), CeresClone:273420 (SEQ ID NO:531), CeresAnnot:8701399 (SEQ ID NO: 533), GI:125540061 (SEQ ID NO:534), GI:115446971 (SEQ ID NO: 535), CeresClone:1802499 (SEQ ID NO:537), CeresClone:1850157 (SEQ ID NO: 539), CeresClone:1471240 (SEQ IDNO: 541), CeresAnnot:8679942 (SEQ ID NO: 543), CeresClone:1024049 (SEQID NO: 545), CeresAnnot:885518 (SEQ ID NO: 547), CeresAnnot:871243 (SEQID NO: 549), CeresAnnot:1461629 (SEQ ID NO: 551), GI:27754556 (SEQ IDNO: 552), CeresAnnot:8679941 (SEQ ID NO: 554), CeresClone:1846767 (SEQID NO: 556), GI:118489467 (SEQ ID NO: 557), and CeresAnnot:1480319 (SEQID NO: 559). In some cases, a functional homolog of SEQ ID NO: 483 hasan amino acid sequence with at least 45% sequence identity, e.g., 50%,52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%sequence identity, to the amino acid sequence set forth in SEQ ID NO:483. In some cases, a functional homolog of SEQ ID NO: 483 has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to one or more functional homologs of SEQ ID NO: 483 describedabove or set forth in the Sequence Listing.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 562 are provided in FIG. 2 and inthe Sequence Listing. Such functional homologs include, for example,CeresAnnot:8703443 (SEQ ID NO: 564), GI:194702514 (SEQ ID NO: 565),CeresClone:699934 (SEQ ID NO: 567), GI:32488374 (SEQ ID NO: 568),CeresClone:1642517 (SEQ ID NO: 570), CeresClone:1799746 (SEQ ID NO:572), GI:224077486 (SEQ ID NO: 573), GI:83283997 (SEQ ID NO: 574),GI:171451994 (SEQ ID NO: 575), GI:15223416 (SEQ ID NO: 576),CeresClone:1999925 (SEQ ID NO: 578), CeresClone:100177220 (SEQ ID NO:580), CeresClone:1822001 (SEQ ID NO: 582), CeresClone:570418 (SEQ ID NO:584), CeresClone:1998324 (SEQ ID NO: 586), CeresClone:706252 (SEQ ID NO:588), GI:77554837 (SEQ ID NO: 589), GI:125536425 (SEQ ID NO: 590),CeresAnnot:1447508 (SEQ ID NO: 592), CeresClone:1965618 (SEQ ID NO:594), CeresClone:1626139 (SEQ ID NO: 596), CeresAnnot:8640237 (SEQ IDNO: 598), GI:115450453 (SEQ ID NO: 599), CeresAnnot:1438634 (SEQ ID NO:601), GI:147787209 (SEQ ID NO: 602), GI:115483110 (SEQ ID NO: 603),CeresClone:263964 (SEQ ID NO: 605), CeresAnnot:1449592 (SEQ ID NO: 607),GI:115461178 (SEQ ID NO: 608), GI:29124977 (SEQ ID NO: 609),CeresClone:476087 (SEQ ID NO: 611), CeresClone:1587840 (SEQ ID NO: 613),CeresClone:1808797 (SEQ ID NO: 615), CeresClone:538771 (SEQ ID NO: 617),CeresClone:1851138 (SEQ ID NO: 619), CeresClone:1049645 (SEQ ID NO:621), GI:92897781 (SEQ ID NO: 622), CeresAnnot:1487378 (SEQ ID NO: 624),GI:92897782 (SEQ ID NO: 625), CeresClone:648917 (SEQ ID NO: 627),CeresClone:100011205 (SEQ ID NO: 629), GI:116783342 (SEQ ID NO: 630),CeresAnnot:1449591 (SEQ ID NO: 632), CeresClone:521942 (SEQ ID NO: 634),CeresClone:1653508 (SEQ ID NO: 636), and CeresAnnot:1487377 (SEQ ID NO:638). In some cases, a functional homolog of SEQ ID NO: 562 has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 562. Insome cases, a functional homolog of SEQ ID NO: 562 has an amino acidsequence with at least 45% sequence identity, e.g., 50%, 52%, 56%, 59%,61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to one or more functional homologs of SEQ ID NO: 562 describedabove or set forth in the Sequence Listing.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 246 are provided in FIG. 3 and inthe Sequence Listing. Such functional homologs include, for example,CeresClone:1791988 (SEQ ID NO: 248), CeresAnnot:8632546 (SEQ ID NO:250), GI:115455537 (SEQ ID NO: 251), GI:118486821 (SEQ ID NO: 252),CeresClone:537690 (SEQ ID NO: 254), CeresAnnot:880540 (SEQ ID NO: 256),CeresClone:797459 (SEQ ID NO: 258), CeresClone:630408 (SEQ ID NO: 260),GI:125557053 (SEQ ID NO: 261), GI:125588020 (SEQ ID NO: 262),CeresAnnot:1733246 (SEQ ID NO: 264), CeresAnnot:1451294 (SEQ ID NO:266), CeresAnnot:1457031 (SEQ ID NO: 268), CeresClone:100063507 (SEQ IDNO: 270), CeresClone:560820 (SEQ ID NO: 272), CeresClone:1104471 (SEQ IDNO: 274), GI:30690890 (SEQ ID NO: 275), GI:18402692 (SEQ ID NO: 276),and CeresClone:2686 (SEQ ID NO: 278). In some cases, a functionalhomolog of SEQ ID NO: 246 has an amino acid sequence with at least 45%sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acidsequence set forth in SEQ ID NO: 246. In some cases, a functionalhomolog of SEQ ID NO: 246 has an amino acid sequence with at least 45%sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to one or morefunctional homologs of SEQ ID NO: 246 described above or set forth inthe Sequence Listing.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 111 are provided in FIG. 4 and inthe Sequence Listing. Such functional homologs include, for example,CeresAnnot:8726250 (SEQ ID NO: 113), CeresClone:899059 (SEQ ID NO:115),CeresClone:945132 (SEQ ID NO:117), GI:115462673 (SEQ ID NO:118),CeresClone:16400 (SEQ ID NO:120), CeresClone:1712201 (SEQ ID NO:122),CeresAnnot:1524669 (SEQ ID NO:124), CeresAnnot:8672987 (SEQ ID NO:126),CeresClone:1434951 (SEQ ID NO:128), CeresClone:299745 (SEQ ID NO:130),CeresClone:323696 (SEQ ID NO:132), GI:194695666 (SEQ ID NO:133),CeresClone:1771257 (SEQ ID NO:135), GI:115445433 (SEQ ID NO:136),CeresAnnot:8667876 (SEQ ID NO:138), GI:115438957 (SEQ ID NO:139),CeresClone:1100814 (SEQ ID NO:141), CeresClone:1029710 (SEQ ID NO:143),CeresClone:969326 (SEQ ID NO:145), CeresClone:100955392 (SEQ ID NO:147),GI:225454450 (SEQ ID NO:148), GI:116779724 (SEQ ID NO:149),CeresAnnot:1447561 (SEQ ID NO:151), GI:20149060 (SEQ ID NO:152),GI:225462683 (SEQ ID NO:153), and CeresClone:595099 (SEQ ID NO:155). Insome cases, a functional homolog of SEQ ID NO: 111 has an amino acidsequence with at least 45% sequence identity, e.g., 50%, 52%, 56%, 59%,61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 111. Insome cases, a functional homolog of SEQ ID NO: 111 has an amino acidsequence with at least 45% sequence identity, e.g., 50%, 52%, 56%, 59%,61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to one or more functional homologs of SEQ ID NO: 111 describedabove or set forth in the Sequence Listing.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 348 are provided in FIG. 5 and inthe Sequence Listing. Such functional homologs include, for example,CeresAnnot:8642214 (SEQ ID NO: 350), GI:115451805 (SEQ ID NO: 351),CeresClone:890595 (SEQ ID NO: 353), CeresAnnot:1463701 (SEQ ID NO: 355),CeresClone:1840970 (SEQ ID NO: 357), CeresClone:672495 (SEQ ID NO: 359),GI:225424452 (SEQ ID NO: 360), GI:15223878 (SEQ ID NO: 361), GI:13560781(SEQ ID NO: 362), GI:6681351 (SEQ ID NO: 363), GI:116786783 (SEQ ID NO:364), GI:125543052 (SEQ ID NO: 365), GI:124109193 (SEQ ID NO: 366),CeresAnnot:8653921 (SEQ ID NO: 368), CeresClone:1995976 (SEQ ID NO:370), CeresClone:369312 (SEQ ID NO: 372), GI:17047034 (SEQ ID NO: 373),GI:118482018 (SEQ ID NO: 374), GI:125530964 (SEQ ID NO: 375),GI:125563629 (SEQ ID NO: 376), GI:147797772 (SEQ ID NO: 377),CeresClone:18876 (SEQ ID NO: 379), GI:125540767 (SEQ ID NO: 380),GI:115448069 (SEQ ID NO: 381), CeresClone:683310 (SEQ ID NO: 383),GI:125605601 (SEQ ID NO: 384), CeresClone:1922671 (SEQ ID NO: 386),CeresClone:100961902 (SEQ ID NO: 388), CeresAnnot:1447077 (SEQ ID NO:390), CeresClone:1643790 (SEQ ID NO: 392), GI:125580663 (SEQ ID NO:393), GI:116785331 (SEQ ID NO: 394), CeresAnnot:1485570 (SEQ ID NO:396), CeresAnnot:8681188 (SEQ ID NO: 398), CeresClone:1818189 (SEQ IDNO: 400), CeresClone:100861631 (SEQ ID NO: 402), CeresAnnot:8671232 (SEQID NO: 404), CeresClone:1813525 (SEQ ID NO: 406), GI:15222593 (SEQ IDNO: 407), GI:42795460 (SEQ ID NO: 408), CeresClone:1828819 (SEQ ID NO:410), CeresAnnot:1460297 (SEQ ID NO: 412), GI:225424689 (SEQ ID NO:413), and GI:76786474 (SEQ ID NO: 414). In some cases, a functionalhomolog of SEQ ID NO: 348 has an amino acid sequence with at least 45%sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acidsequence set forth in SEQ ID NO: 348. In some cases, a functionalhomolog of SEQ ID NO: 348 has an amino acid sequence with at least 45%sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to one or morefunctional homologs of SEQ ID NO: 348 described above or set forth inthe Sequence Listing.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 774 are provided in FIG. 6 and inthe Sequence Listing. Such functional homologs include, for example,GI:115483997 (SEQ ID NO: 775), GI:13398414 (SEQ ID NO: 776), GI:33151175(SEQ ID NO: 777), GI:119507455 (SEQ ID NO: 778), CeresClone:549408 (SEQID NO: 780), GI:37777015 (SEQ ID NO: 781), GI:157313302 (SEQ ID NO:782), GI:157072586 (SEQ ID NO: 783), CeresAnnot:1506572 (SEQ ID NO:785), GI:16417958 (SEQ ID NO: 786), CeresAnnot:556941 (SEQ ID NO: 788),GI:225440254 (SEQ ID NO: 789), CeresClone:1753603 (SEQ ID NO: 791),CeresClone:236733 (SEQ ID NO: 793), CeresClone:1786359 (SEQ ID NO: 795),GI:115487150 (SEQ ID NO: 796), CeresAnnot:8682811 (SEQ ID NO: 798),GI:13398412 (SEQ ID NO: 799), GI:116310992 (SEQ ID NO: 800), GI:38347003(SEQ ID NO: 801), GI:116739148 (SEQ ID NO: 802), GI:22324432 (SEQ ID NO:803), CeresAnnot:1453426 (SEQ ID NO: 805), CeresAnnot:8657414 (SEQ IDNO: 807), GI:108707861 (SEQ ID NO: 808), CeresAnnot:1528070 (SEQ ID NO:810), GI:22327075 (SEQ ID NO: 811), GI:50507838 (SEQ ID NO: 812),GI:168060089 (SEQ ID NO: 813), GI:160890886 (SEQ ID NO: 814),GI:189464007 (SEQ ID NO: 815), GI:154492683 (SEQ ID NO: 816),GI:146300858 (SEQ ID NO: 817), GI:150008552 (SEQ ID NO: 818),GI:86142284 (SEQ ID NO: 819), GI:148269769 (SEQ ID NO: 820), andGI:170288456 (SEQ ID NO: 821). In some cases, a functional homolog ofSEQ ID NO: 774 has an amino acid sequence with at least 45% sequenceidentity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence setforth in SEQ ID NO: 774. In some cases, a functional homolog of SEQ IDNO: 774 has an amino acid sequence with at least 45% sequence identity,e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,98%, or 99% sequence identity, to one or more functional homologs of SEQID NO: 774 described above or set forth in the Sequence Listing.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 416 are provided in FIG. 7 and inthe Sequence Listing. Such functional homologs include, for example,CeresAnnot:8656625 (SEQ ID NO: 418), GI:162462515 (SEQ ID NO: 419),GI:75133694 (SEQ ID NO: 420), CeresClone:829440 (SEQ ID NO: 422),GI:118488472 (SEQ ID NO: 423), GI:90657534 (SEQ ID NO: 424),CeresClone:1237946 (SEQ ID NO: 426), GI:225456557 (SEQ ID NO: 427),CeresAnnot:1355066 (SEQ ID NO: 429), GI:38194917 (SEQ ID NO: 430),GI:116788824 (SEQ ID NO: 431), CeresClone:1848658 (SEQ ID NO: 433),GI:116790012 (SEQ ID NO: 434), CeresClone:570485 (SEQ ID NO: 436),GI:125559102 (SEQ ID NO: 437), CeresClone:1957107 (SEQ ID NO: 439),CeresClone:1781794 (SEQ ID NO: 441), GI:115453531 (SEQ ID NO: 442),CeresClone:285169 (SEQ ID NO: 444), CeresAnnot:1450186 (SEQ ID NO: 446),CeresClone:1806851 (SEQ ID NO: 448), GI:38194916 (SEQ ID NO: 449),GI:225451792 (SEQ ID NO: 450), GI:225456559 (SEQ ID NO: 451),GI:224124236 (SEQ ID NO: 452), CeresClone:17250 (SEQ ID NO: 454),CeresAnnot:1363625 (SEQ ID NO: 456), CeresAnnot:1450185 (SEQ ID NO:458), GI:125552171 (SEQ ID NO: 459), GI:115463639 (SEQ ID NO: 460),CeresAnnot:1809854 (SEQ ID NO: 462), GI:162462330 (SEQ ID NO: 463),CeresAnnot:1326475 (SEQ ID NO: 465), GI:125559101 (SEQ ID NO: 466),CeresAnnot:8632643 (SEQ ID NO: 468), CeresClone:1546455 (SEQ ID NO:470), CeresClone:1788775 (SEQ ID NO: 472), GI:162462156 (SEQ ID NO:473), GI:125545759 (SEQ ID NO: 474), CeresClone:236876 (SEQ ID NO: 476),CeresAnnot:8640602 (SEQ ID NO: 478), GI:30090032 (SEQ ID NO: 479),GI:38230578 (SEQ ID NO: 480), and GI:115453533 (SEQ ID NO: 481). In somecases, a functional homolog of SEQ ID NO: 416 has an amino acid sequencewith at least 45% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to theamino acid sequence set forth in SEQ ID NO: 416. In some cases, afunctional homolog of SEQ ID NO: 416 has an amino acid sequence with atleast 45% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to one ormore functional homologs of SEQ ID NO: 416 described above or set forthin the Sequence Listing.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 2 are provided in FIG. 8 and in theSequence Listing. Such functional homologs include, for example,CeresAnnot:8701928 (SEQ ID NO: 4), CeresClone:630287 (SEQ ID NO: 6),GI:115447391 (SEQ ID NO: 7), GI:225453032 (SEQ ID NO: 8),CeresClone:1919301 (SEQ ID NO: 10), CeresAnnot:883070 (SEQ ID NO: 12),CeresAnnot:1469624 (SEQ ID NO: 14), GI:168065791 (SEQ ID NO: 15),CeresClone:1887777 (SEQ ID NO: 17), GI:57834149 (SEQ ID NO: 18),GI:116310214 (SEQ ID NO: 19), GI:18087513 (SEQ ID NO: 20), GI:147841543(SEQ ID NO: 21), GI:168014382 (SEQ ID NO: 22), and CeresAnnot:8462062(SEQ ID NO: 24). In some cases, a functional homolog of SEQ ID NO: 2 hasan amino acid sequence with at least 45% sequence identity, e.g., 50%,52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%sequence identity, to the amino acid sequence set forth in SEQ ID NO: 2.In some cases, a functional homolog of SEQ ID NO: 2 has an amino acidsequence with at least 45% sequence identity, e.g., 50%, 52%, 56%, 59%,61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to one or more functional homologs of SEQ ID NO: 2 describedabove or set forth in the Sequence Listing.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 157 are provided in FIG. 9 and inthe Sequence Listing. Such functional homologs include, for example,GI:56409850 (SEQ ID NO: 158), CeresAnnot:8740887 (SEQ ID NO: 160),GI:162460428 (SEQ ID NO: 161), GI:115453815 (SEQ ID NO: 162),GI:56409844 (SEQ ID NO: 163), GI:31339690 (SEQ ID NO: 164), GI:9294073(SEQ ID NO: 165), CeresAnnot:1473325 (SEQ ID NO: 167), GI:31296713 (SEQID NO: 168), CeresClone:1925376 (SEQ ID NO: 170), GI:56409848 (SEQ IDNO: 171), GI:125544555 (SEQ ID NO: 172), GI:115445881 (SEQ ID NO: 173),CeresAnnot:8674833 (SEQ ID NO: 175), CeresClone:914572 (SEQ ID NO: 177),CeresAnnot:8659084 (SEQ ID NO: 179), CeresClone:1781320 (SEQ ID NO:181), GI:53791307 (SEQ ID NO: 182), CeresAnnot:8659080 (SEQ ID NO: 184),GI:212275650 (SEQ ID NO: 185), CeresClone:1818693 (SEQ ID NO: 187),CeresClone:508386 (SEQ ID NO: 189), GI:53791309 (SEQ ID NO: 190),CeresAnnot:8659051 (SEQ ID NO: 192), CeresClone:1862153 (SEQ ID NO:194), CeresClone:1902844 (SEQ ID NO: 196), GI:212275101 (SEQ ID NO:197), CeresClone:1844210 (SEQ ID NO: 199), CeresAnnot:8658929 (SEQ IDNO: 201), GI:125555301 (SEQ ID NO: 202), CeresClone:825530 (SEQ ID NO:204), GI:115444075 (SEQ ID NO: 205), CeresClone:1748522 (SEQ ID NO:207), GI:115445889 (SEQ ID NO: 208), CeresAnnot:8671335 (SEQ ID NO:210), GI:53791308 (SEQ ID NO: 211), CeresClone:1899806 (SEQ ID NO: 213),CeresClone:1726616 (SEQ ID NO: 215), GI:162460449 (SEQ ID NO: 216),CeresClone:1770027 (SEQ ID NO: 218), CeresAnnot:1467806 (SEQ ID NO:220), GI:55792425 (SEQ ID NO: 221), GI:56409862 (SEQ ID NO: 222),GI:115482674 (SEQ ID NO: 223), CeresClone:815962 (SEQ ID NO: 225),GI:56409860 (SEQ ID NO: 226), CeresAnnot:8670072 (SEQ ID NO: 228),CeresAnnot:1473327 (SEQ ID NO: 230), CeresClone:1726182 (SEQ ID NO:232), CeresAnnot:8734902 (SEQ ID NO: 234), CeresAnnot:8741882 (SEQ IDNO: 236), CeresClone:761431 (SEQ ID NO: 238), CeresAnnot:8678791 (SEQ IDNO: 240), CeresClone:845464 (SEQ ID NO: 242), and CeresClone:1726076(SEQ ID NO: 244). In some cases, a functional homolog of SEQ ID NO: 157has an amino acid sequence with at least 45% sequence identity, e.g.,50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or99% sequence identity, to the amino acid sequence set forth in SEQ IDNO: 157. In some cases, a functional homolog of SEQ ID NO: 157 has anamino acid sequence with at least 45% sequence identity, e.g., 50%, 52%,56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%sequence identity, to one or more functional homologs of SEQ ID NO: 157described above or set forth in the Sequence Listing.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 280 are provided in FIG. 10 and inthe Sequence Listing. Such functional homologs include, for example,CeresAnnot:8681689 (SEQ ID NO: 282), GI:226529851 (SEQ ID NO: 283),GI:115448865 (SEQ ID NO: 284), GI:154163107 (SEQ ID NO: 285),GI:147817757 (SEQ ID NO: 286), CeresClone:1925709 (SEQ ID NO: 288),GI:15227566 (SEQ ID NO: 289), GI:20138107 (SEQ ID NO: 290),CeresClone:934069 (SEQ ID NO: 292), CeresAnnot:8681691 (SEQ ID NO: 294),GI:46805726 (SEQ ID NO: 295), GI:125541250 (SEQ ID NO: 296),GI:115448869 (SEQ ID NO: 297), GI:115467048 (SEQ ID NO: 298),GI:51090521 (SEQ ID NO:299), GI:125554524 (SEQ ID NO:300),CeresAnnot:8735787 (SEQ ID NO:302), GI:15227563 (SEQ ID NO:303),CeresAnnot:8681690 (SEQ ID NO:305), GI:15223062 (SEQ ID NO:306),CeresAnnot:8735782 (SEQ ID NO:308), GI:115467046 (SEQ ID NO:309),GI:125554519 (SEQ ID NO:310), GI:125596466 (SEQ ID NO:311), GI:20138442(SEQ ID NO:312), CeresAnnot:1448326 (SEQ ID NO:314), CeresAnnot:8735776(SEQ ID NO:316), GI:125554515 (SEQ ID NO:317), GI:154163097 (SEQ IDNO:318), CeresAnnot:8673445 (SEQ ID NO:320), GI:115445521 (SEQ IDNO:321), CeresAnnot:1448328 (SEQ ID NO:323), CeresAnnot:1437779 (SEQ IDNO:325), GI:15226507 (SEQ ID NO:326), GI:154163099 (SEQ ID NO:327),GI:93139696 (SEQ ID NO:328), GI:154163101 (SEQ ID NO:329),CeresAnnot:1448327 (SEQ ID NO:331), CeresAnnot:8681687 (SEQ ID NO:333),CeresAnnot:1437782 (SEQ ID NO:335), GI:20138443 (SEQ ID NO:336),GI:15226501 (SEQ ID NO:337), GI:125541240 (SEQ ID NO:338), GI:115458656(SEQ ID NO:339), GI:125548499 (SEQ ID NO:340), (CeresAnnot:8654550SEQ IDNO:342), CeresAnnot:8701112 (SEQ ID NO:344), and CeresClone:1530993 (SEQID NO:346). In some cases, a functional homolog of SEQ ID NO: 280 has anamino acid sequence with at least 45% sequence identity, e.g., 50%, 52%,56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%sequence identity, to the amino acid sequence set forth in SEQ ID NO:280. In some cases, a functional homolog of SEQ ID NO: 280 has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to one or more functional homologs of SEQ ID NO: 280 describedabove or set forth in the Sequence Listing.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 641 are provided in FIG. 11 and inthe Sequence Listing. Such functional homologs include, for example,CeresAnnot:8744420 (SEQ ID NO: 643), CeresClone:331385 (SEQ ID NO: 645),GI:115469712 (SEQ ID NO:646), GI:1890577 (SEQ ID NO:647), GI:51039064(SEQ ID NO:648), GI:14330332 (SEQ ID NO:649), GI:147854712 (SEQ IDNO:650), GI:157352236 (SEQ ID NO:651), GI:118722746 (SEQ ID NO:652),GI:8886867 (SEQ ID NO:653), GI:115334952 (SEQ ID NO:654),CeresClone:1789502 (SEQ ID NO:656), CeresClone:1805428 (SEQ ID NO:658),CeresClone:1724099 (SEQ ID NO:660), CeresClone:1724817 (SEQ ID NO:662),CeresClone:1804995 (SEQ ID NO:664), CeresClone:1446366 (SEQ ID NO:666),CeresClone:1054422 (SEQ ID NO:668), CeresClone:263803 (SEQ ID NO:670),CeresClone:1821034 (SEQ ID NO:672), CeresClone:1806021 (SEQ ID NO:674),CeresClone:1727689 (SEQ ID NO:676), GI:115469720 (SEQ ID NO:677),CeresAnnot:8744425 (SEQ ID NO:679), GI:212275237 (SEQ ID NO:680),CeresClone:1724271 (SEQ ID NO:682), CeresClone:247073 (SEQ ID NO:684),CeresClone:1020658 (SEQ ID NO:686), GI:1890575 (SEQ ID NO:687),GI:225446111 (SEQ ID NO:688), GI:225446115 (SEQ ID NO:689), GI:147854714(SEQ ID NO:690), GI:68532877 (SEQ ID NO:691), GI:147779866 (SEQ IDNO:692), CeresClone:100062911 (SEQ ID NO:694), GI:225446117 (SEQ IDNO:695), CeresClone:1832719 (SEQ ID NO:697), CeresClone:1793297 (SEQ IDNO:699), CeresClone:1848637 (SEQ ID NO:701), GI:225446103 (SEQ IDNO:702), CeresAnnot:1362908 (SEQ ID NO:704), CeresClone:100064069 (SEQID NO:706), CeresAnnot:1469128 (SEQ ID NO:708), CeresClone:656868 (SEQID NO:710), CeresClone:1793334 (SEQ ID NO:712), GI:29500891 (SEQ IDNO:713), CeresClone:1895226 (SEQ ID NO:715), GI:8886865 (SEQ ID NO:716),CeresAnnot:878947 (SEQ ID NO:718), CeresClone:1045431 (SEQ ID NO:720),GI:22947852 (SEQ ID NO:721), CeresClone:1855067 (SEQ ID NO:723),GI:17064792 (SEQ ID NO:724), CeresClone:662227 (SEQ ID NO:726),GI:225446109 (SEQ ID NO:727), CeresClone:522574 (SEQ ID NO:729),GI:115334954 (SEQ ID NO:730), CeresClone:581426 (SEQ ID NO:732),GI:124109191 (SEQ ID NO:733), CeresAnnot:1471882 (SEQ ID NO:735),GI:34809190 (SEQ ID NO:736), GI:29500893 (SEQ ID NO:737),CeresAnnot:1452398 (SEQ ID NO:739), GI:124109199 (SEQ ID NO:740),CeresAnnot:1478206 (SEQ ID NO:742), CeresAnnot:1445599 (SEQ ID NO:744),CeresAnnot:1452397 (SEQ ID NO:746), GI:19911573 (SEQ ID NO:747),GI:124109181 (SEQ ID NO:748), GI:22327914 (SEQ ID NO:749), GI:42795468(SEQ ID NO:750), GI:42795462 (SEQ ID NO:751), CeresAnnot:1466060 (SEQ IDNO:753), CeresAnnot:8461207 (SEQ ID NO:755), CeresAnnot:1506985 (SEQ IDNO:757), GI:3901012 (SEQ ID NO:758), CeresAnnot:1443040 (SEQ ID NO:760),GI:90811697 (SEQ ID NO:761), CeresAnnot:1443041 (SEQ ID NO:763),GI:157358970 (SEQ ID NO:764), GI:90656516 (SEQ ID NO:765), GI:577066(SEQ ID NO:766), GI:90656520 (SEQ ID NO:767), GI:88683124 (SEQ IDNO:768), GI:90656518 (SEQ ID NO:769), CeresAnnot:1482565 (SEQ IDNO:771), GI:15238891 (SEQ ID NO:772), and Ceres Clone ID No. 933491 (SEQID NO: 823). In some cases, a functional homolog of SEQ ID NO: 641 hasan amino acid sequence with at least 45% sequence identity, e.g., 50%,52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%sequence identity, to the amino acid sequence set forth in SEQ ID NO:641. In some cases, a functional homolog of SEQ ID NO: 641 has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to one or more functional homologs of SEQ ID NO: 641 describedabove or set forth in the Sequence Listing.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 26 are provided in FIG. 12 and inthe Sequence Listing.

Such functional homologs include, for example, CeresClone:570179 (SEQ IDNO: 28), GI:54290293 (SEQ ID NO:29), GI:1617121 (SEQ ID NO:30),CeresAnnot:8724383 (SEQ ID NO:32), CeresClone:896724 (SEQ ID NO:34),CeresClone:607452 (SEQ ID NO:36), GI:37904392 (SEQ ID NO:37),CeresClone:1870473 (SEQ ID NO:39), CeresClone:2026564 (SEQ ID NO:41),CeresClone:2004365 (SEQ ID NO:43), CeresClone:2020677 (SEQ ID NO:45),CeresClone:2039538 (SEQ ID NO:47), CeresClone:844611 (SEQ ID NO:49),GI:125526847 (SEQ ID NO:50), CeresClone:597887 (SEQ ID NO:52),GI:58396949 (SEQ ID NO:53), CeresClone:684778 (SEQ ID NO:55),CeresClone:699511 (SEQ ID NO:57), CeresClone:1803377 (SEQ ID NO:59),CeresClone:1888961 (SEQ ID NO:61), CeresClone:897331 (SEQ ID NO:63),CeresClone:617775 (SEQ ID NO:65), GI:20513866 (SEQ ID NO:66),CeresAnnot:8724387 (SEQ ID NO:68), CeresClone:1804405 (SEQ ID NO:70),GI:48093396 (SEQ ID NO:71), GI:108862602 (SEQ ID NO:72), GI:115488400(SEQ ID NO:73), CeresClone:759663 (SEQ ID NO:75), CeresClone:1801827(SEQ ID NO:77), GI:48093418 (SEQ ID NO:78), GI:48093360 (SEQ ID NO:79),CeresClone:1457620 (SEQ ID NO:81), GI:48093370 (SEQ ID NO:82),CeresClone:639183 (SEQ ID NO:84), CeresClone:1453564 (SEQ ID NO:86),CeresClone:1531954 (SEQ ID NO:88), CeresClone:1460371 (SEQ ID NO:90),CeresClone:1627479 (SEQ ID NO:92), CeresClone:992630 (SEQ ID NO:94),CeresClone:685480 (SEQ ID NO:96), GI:75994159 (SEQ ID NO:97),CeresAnnot:8724380 (SEQ ID NO:99), GI:48093378 (SEQ ID NO:100),GI:75994143 (SEQ ID NO:101), GI:75994153 (SEQ ID NO:102),CeresAnnot:8724381 (SEQ ID NO:104), GI:75994157 (SEQ ID NO:105),CeresClone:730301 (SEQ ID NO:107), and CeresAnnot:8724388 (SEQ IDNO:109). In some cases, a functional homolog of SEQ ID NO: 26 has anamino acid sequence with at least 45% sequence identity, e.g., 50%, 52%,56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%sequence identity, to the amino acid sequence set forth in SEQ ID NO:26. In some cases, a functional homolog of SEQ ID NO: 26 has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to one or more functional homologs of SEQ ID NO: 26 describedabove or set forth in the Sequence Listing.

The identification of conserved regions in a biomasscomposition-modulating polypeptide facilitates production of variants ofbiomass composition-modulating polypeptides. Variants of biomasscomposition-modulating polypeptides typically have 10 or fewerconservative amino acid substitutions within the primary amino acidsequence, e.g., 7 or fewer conservative amino acid substitutions, 5 orfewer conservative amino acid substitutions, or between 1 and 5conservative substitutions. A useful variant polypeptide can beconstructed based on one of the alignments set forth in FIG. 1 , FIG. 2, FIG. 3 , FIG. 4 , FIG. 5 , FIG. 6 , FIG. 7 , FIG. 8 , FIG. 9 , FIG. 10, FIG. 11 , or FIG. 12 , and/or homologs identified in the SequenceListing. Such a polypeptide includes the conserved regions, arranged inthe order depicted in the Figure from amino-terminal end tocarboxy-terminal end. Such a polypeptide may also include zero, one, ormore than one amino acid in positions marked by dashes. When no aminoacids are present at positions marked by dashes, the length of such apolypeptide is the sum of the amino acid residues in all conservedregions. When amino acids are present at a position marked by dashes,such a polypeptide has a length that is the sum of the amino acidresidues in all conserved regions and all dashes.

C. Functional Homologs Identified by HMMER

In some embodiments, useful biomass composition-modulating polypeptidesinclude those that fit a Hidden Markov Model based on the polypeptidesset forth in any one of FIGS. 1-12 . A Hidden Markov Model (HMM) is astatistical model of a consensus sequence for a group of functionalhomologs. See, Durbin et al., Biological Sequence Analysis:Probabilistic Models of Proteins and Nucleic Acids, Cambridge UniversityPress, Cambridge, UK (1998). An HMM is generated by the program HMMER2.3.2 with default program parameters, using the sequences of the groupof functional homologs as input. The multiple sequence alignment isgenerated by ProbCons (Do et al., Genome Res., 15(2):330-40 (2005))version 1.11 using a set of default parameters: -c,—consistency REPS of2; -ir,—iterative-refinement REPS of 100; -pre,—pre-training REPS of 0.ProbCons is a public domain software program provided by StanfordUniversity.

The default parameters for building an HMM (hmmbuild) are as follows:the default “architecture prior” (archpri) used by MAP architectureconstruction is 0.85, and the default cutoff threshold (idlevel) used todetermine the effective sequence number is 0.62. HMMER 2.3.2 wasreleased Oct. 3, 2003 under a GNU general public license, and isavailable from various sources on the World Wide Web such ashmmer.janelia.org; hmmer wustl.edu; and fr.com/hmmer232/. Hmmbuildoutputs the model as a text file.

The HMM for a group of functional homologs can be used to determine thelikelihood that a candidate biomass composition-modulating polypeptidesequence is a better fit to that particular HMM than to a null HMMgenerated using a group of sequences that are not structurally orfunctionally related. The likelihood that a candidate polypeptidesequence is a better fit to an HMM than to a null HMM is indicated bythe HMM bit score, a number generated when the candidate sequence isfitted to the HMM profile using the HMMER hmmsearch program. Thefollowing default parameters are used when running hmmsearch: thedefault E-value cutoff (E) is 10.0, the default bit score cutoff (T) isnegative infinity, the default number of sequences in a database (Z) isthe real number of sequences in the database, the default E-value cutofffor the per-domain ranked hit list (domE) is infinity, and the defaultbit score cutoff for the per-domain ranked hit list (domT) is negativeinfinity. A high HMM bit score indicates a greater likelihood that thecandidate sequence carries out one or more of the biochemical orphysiological function(s) of the polypeptides used to generate the HMM.A high HMM bit score is at least 20, and often is higher. Slightvariations in the HMM bit score of a particular sequence can occur dueto factors such as the order in which sequences are processed foralignment by multiple sequence alignment algorithms such as the ProbConsprogram. Nevertheless, such HMM bit score variation is minor.

The biomass composition-modulating polypeptides discussed below fit theindicated HMM with an HMM bit score greater than to 65. (e.g., greaterthan 70, 80, 90, 100, 120, 140, 200, 300, 500, 1000, 1500, or 2000). Insome embodiments, the HMM bit score of a biomass composition-modulatingpolypeptide discussed below is about 50%, 60%, 70%, 80%, 90%, or 95% ofthe HMM bit score of a functional homolog provided in the SequenceListing of this application. In some embodiments, a biomasscomposition-modulating polypeptide discussed below fits the indicatedHMM with an HMM bit score greater than 210, and has a domain indicativeof a biomass composition-modulating polypeptide. In some embodiments, abiomass composition-modulating polypeptide discussed below fits theindicated HMM with an HMM bit score greater than 210, and has 65% orgreater sequence identity (e.g., 75%, 80%, 85%, 90%, 95%, or 100%sequence identity) to an amino acid sequence shown in any one of FIGS.1-12 .

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 84 (e.g., greater than 100, 120, 140, 160, 180,200, 220, 240, 250, 260, 270, 280, or 290) when fitted to an HMMgenerated from the amino acid sequences set forth in FIG. 1 areidentified in the Sequence Listing of this application. Suchpolypeptides include, for example, SEQ ID NOs: 483, 485, 486, 488, 490,492, 493, 495, 497, 498, 500, 501, 503, 504, 506, 508, 509, 510, 512,514, 515, 516, 517, 519, 521, 522, 523, 525, 527, 529, 531, 533, 534,535, 537, 539, 541, 543, 545, 547, 549, 551, 552, 554, 556, 557, and559.

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 120 (e.g., greater than 125, 130, 140, 150, 160,170, 180, 200, 220, 240, 260, 280, 300, or 315) when fitted to an HMMgenerated from the amino acid sequences set forth in FIG. 2 areidentified in the Sequence Listing of this application. Suchpolypeptides include, for example, SEQ ID NOs: 562, 564, 565, 567, 568,570, 572, 573, 574, 575, 576, 578, 580, 582, 584, 586, 588, 589, 590,592, 594, 596, 598, 599, 601, 602, 603, 605, 607, 608, 609, 611, 613,615, 617, 619, 621, 622, 624, 625, 627, 629, 630, 632, 634, 636, and638.

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 200 (e.g., greater than 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 975, or 1000) whenfitted to an HMM generated from the amino acid sequences set forth inFIG. 3 are identified in the Sequence Listing of this application. Suchpolypeptides include, for example, SEQ ID NOs: 246, 248, 250, 251, 252,254, 256, 258, 260, 261, 262, 264, 266, 268, 270, 272, 274, 275, 276,and 278.

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 93 (e.g., greater than 95, 100, 105, 110, 115,120, 125, 130, 135, 140, or 145) when fitted to an HMM generated fromthe amino acid sequences set forth in FIG. 4 are identified in theSequence Listing of this application. Such polypeptides include, forexample, SEQ ID NOs: 111, 113, 115, 117, 118, 120, 122, 124, 126, 128,130, 132, 133, 135, 136, 138, 139, 141, 143, 145, 147, 148, 149, 151,152, 153, and 155.

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 387 (e.g., greater than 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, or 920) when fitted to an HMM generatedfrom the amino acid sequences set forth in FIG. 5 are identified in theSequence Listing of this application. Such polypeptides include, forexample, SEQ ID NOs: 348, 350, 351, 353, 355, 357, 359, 360, 361, 362,363, 364, 365, 366, 368, 370, 372, 373, 374, 375, 376, 377, 379, 380,381, 383, 384, 386, 388, 390, 392, 393, 394, 396, 398, 400, 402, 404,406, 407, 408, 410, 412, 413, and 414.

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 315 (e.g., greater than 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200,1250, 1300, 1350, 1400, 1450, 1500, 1500, 1550, 1600, 1620, 1630, or1640) when fitted to an HMM generated from the amino acid sequences setforth in FIG. 6 are identified in the Sequence Listing of thisapplication. Such polypeptides include, for example, SEQ ID NOs: 774,775, 776, 777, 778, 780, 781, 782, 783, 785, 786, 788, 789, 791, 793,795, 796, 798, 799, 800, 801, 802, 803, 805, 807, 808, 810, 811, 812,813, 814, 815, 816, 817, 818, 819, 820, and 821.

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 914 (e.g., greater than 920, 940, 960, 980,1000, 1020, 1040,1060, 1080, 1090, or 1100) when fitted to an HMMgenerated from the amino acid sequences set forth in FIG. 7 areidentified in the Sequence Listing of this application. Suchpolypeptides include, for example, SEQ ID NOs: 416, 418, 419, 420, 422,423, 424, 426, 427, 429, 430, 431, 433, 434, 436, 437, 439, 441, 442,444, 446, 448, 449, 450, 451, 452, 454, 456, 458, 459, 460, 462, 463,465, 466, 468, 470, 472, 473, 474, 476, 478, 479, 480, and 481.

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 659 (e.g., greater than 675, 700, 800, 900,1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1425, or 1440)when fitted to an HMM generated from the amino acid sequences set forthin FIG. 8 are identified in the Sequence Listing of this application.Such polypeptides include, for example, SEQ ID NOs: 2, 4, 6, 7, 8, 10,12, 14, 15, 17, 18, 19, 20, 21, 22, and 24. In some embodiments, an HMMcan be generated based on the amino acid sequences set forth in FIG. 8that are truncated at about residue 142.

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 406 (e.g., greater than 420, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,1300, 1350, 1400, 1420, or 1440) when fitted to an HMM generated fromthe amino acid sequences set forth in FIG. 9 are identified in theSequence Listing of this application. Such polypeptides include, forexample, SEQ ID NOs: 157, 158, 160, 161, 162, 163, 164, 165, 167, 168,170, 171, 172, 173, 175, 177, 179, 181, 182, 184, 185, 187, 189, 190,192, 194, 196, 197, 199, 201, 202, 204, 205, 207, 208, 210, 211, 213,215, 216, 218, 220, 221, 222, 223, 225, 226, 228, 230, 232, 234, 236,238, 240, 242, and 244.

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 640 (e.g., greater than 650, 700, 750, 800, 850,900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500, or 1510), when fitted to an HMM generated from the amino acidsequences set forth in FIG. 10 are identified in the Sequence Listing ofthis application. Such polypeptides include, for example, SEQ ID NOs:280, 282, 283, 284, 285, 286, 288, 289, 290, 292, 294, 295, 296, 297,298, 299, 300, 302, 303, 305, 306, 308, 309, 310, 311, 312, 314, 316,317, 318, 320, 321, 323, 325, 326, 327, 328, 329, 331, 333, 335, 336,337, 338, 339, 340, 342, 344, and 346.

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 234 (e.g., greater than 250, 275, 300, 325, 350,375, 400, 424, 450, 475, 500, 525, 550, 575, 600, 626, 650, 675, 700, or720) when fitted to an HMM generated from the amino acid sequences setforth in FIG. 11 are identified in the Sequence Listing of thisapplication. Such polypeptides include, for example, SEQ ID NOs: 641,643, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 656, 658, 660,662, 664, 666, 668, 670, 672, 674, 676, 677, 679, 680, 682, 684, 686,687, 688, 689, 690, 691, 692, 694, 695, 697, 699, 701, 702, 704, 706,708, 710, 712, 713, 715, 716, 718, 720, 721, 723, 724, 726, 727, 729,730, 732, 733, 735, 736, 737, 739, 740, 742, 744, 746, 747, 748, 749,750, 751, 753, 755, 757, 758, 760, 761, 763, 764, 765, 766, 767, 768,769, 771, 772, and 823.

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 131 (e.g., greater than 135, 140, 145, 150, 151,152, 153, or 154) when fitted to an HMM generated from the amino acidsequences set forth in FIG. 12 are identified in the Sequence Listing ofthis application. Such polypeptides include, for example, SEQ ID NOs:26, 28, 29, 30, 32, 34, 36, 37, 39, 41, 43, 45, 47, 49, 50, 52, 53, 55,57, 59, 61, 63, 65, 66, 68, 70, 71, 72, 73, 75, 77, 78, 79, 81, 82, 84,86, 88, 90, 92, 94, 96, 97, 99, 100, 101, 102, 104, 105, 107, and 109.

D. Percent Identity

In some embodiments, a biomass composition-modulating polypeptide has anamino acid sequence with at least 45% sequence identity, e.g., 50%, 52%,56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%sequence identity, to one of the amino acid sequences set forth in SEQID NOs: 2, 4, 6, 7, 8, 10, 12, 14, 15, 17, 18, 19, 20, 21, 22, 24, 26,28, 29, 30, 32, 34, 36, 37, 39, 41, 43, 45, 47, 49, 50, 52, 53, 55, 57,59, 61, 63, 65, 66, 68, 70, 71, 72, 73, 75, 77, 78, 79, 81, 82, 84, 86,88, 90, 92, 94, 96, 97, 99, 100, 101, 102, 104, 105, 107, 109, 111, 113,115, 117, 118, 120, 122, 124, 126, 128, 130, 132, 133, 135, 136, 138,139, 141, 143, 145, 147, 148, 149, 151, 152, 153, 155, 157, 158, 160,161, 162, 163, 164, 165, 167, 168, 170, 171, 172, 173, 175, 177, 179,181, 182, 184, 185, 187, 189, 190, 192, 194, 196, 197, 199, 201, 202,204, 205, 207, 208, 210, 211, 213, 215, 216, 218, 220, 221, 222, 223,225, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,251, 252, 254, 256, 258, 260, 261, 262, 264, 266, 268, 270, 272, 274,275, 276, 278, 280, 282, 283, 284, 285, 286, 288, 289, 290, 292, 294,295, 296, 297, 298, 299, 300, 302, 303, 305, 306, 308, 309, 310, 311,312, 314, 316, 317, 318, 320, 321, 323, 325, 326, 327, 328, 329, 331,333, 335, 336, 337, 338, 339, 340, 342, 344, 346, 348, 350, 351, 353,355, 357, 359, 360, 361, 362, 363, 364, 365, 366, 368, 370, 372, 373,374, 375, 376, 377, 379, 380, 381, 383, 384, 386, 388, 390, 392, 393,394, 396, 398, 400, 402, 404, 406, 407, 408, 410, 412, 413, 414, 416,418, 419, 420, 422, 423, 424, 426, 427, 429, 430, 431, 433, 434, 436,437, 439, 441, 442, 444, 446, 448, 449, 450, 451, 452, 454, 456, 458,459, 460, 462, 463, 465, 466, 468, 470, 472, 473, 474, 476, 478, 479,480, 481, 483, 485, 486, 488, 490, 492, 493, 495, 497, 498, 500, 501,503, 504, 506, 508, 509, 510, 512, 514, 515, 516, 517, 519, 521, 522,523, 525, 527, 529, 531, 533, 534, 535, 537, 539, 541, 543, 545, 547,549, 551, 552, 554, 556, 557, 559, 562, 564, 565, 567, 568, 570, 572,573, 574, 575, 576, 578, 580, 582, 584, 586, 588, 589, 590, 592, 594,596, 598, 599, 601, 602, 603, 605, 607, 608, 609, 611, 613, 615, 617,619, 621, 622, 624, 625, 627, 629, 630, 632, 634, 636, 638, 641, 643,645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 656, 658, 660, 662,664, 666, 668, 670, 672, 674, 676, 677, 679, 680, 682, 684, 686, 687,688, 689, 690, 691, 692, 694, 695, 697, 699, 701, 702, 704, 706, 708,710, 712, 713, 715, 716, 718, 720, 721, 723, 724, 726, 727, 729, 730,732, 733, 735, 736, 737, 739, 740, 742, 744, 746, 747, 748, 749, 750,751, 753, 755, 757, 758, 760, 761, 763, 764, 765, 766, 767, 768, 769,771, 772, 774, 775, 776, 777, 778, 780, 781, 782, 783, 785, 786, 788,789, 791, 793, 795, 796, 798, 799, 800, 801, 802, 803, 805, 807, 808,810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, and 823.Polypeptides having such a percent sequence identity often have a domainindicative of a biomass composition-modulating polypeptide and/or havean HMM bit score that is greater than 65, as discussed above. Amino acidsequences of biomass composition-modulating polypeptides having at least80% sequence identity to one of the amino acid sequences set forth inSEQ ID NOs: 2, 4, 6, 7, 8, 10, 12, 14, 15, 17, 18, 19, 20, 21, 22, 24,26, 28, 29, 30, 32, 34, 36, 37, 39, 41, 43, 45, 47, 49, 50, 52, 53, 55,57, 59, 61, 63, 65, 66, 68, 70, 71, 72, 73, 75, 77, 78, 79, 81, 82, 84,86, 88, 90, 92, 94, 96, 97, 99, 100, 101, 102, 104, 105, 107, 109, 111,113, 115, 117, 118, 120, 122, 124, 126, 128, 130, 132, 133, 135, 136,138, 139, 141, 143, 145, 147, 148, 149, 151, 152, 153, 155, 157, 158,160, 161, 162, 163, 164, 165, 167, 168, 170, 171, 172, 173, 175, 177,179, 181, 182, 184, 185, 187, 189, 190, 192, 194, 196, 197, 199, 201,202, 204, 205, 207, 208, 210, 211, 213, 215, 216, 218, 220, 221, 222,223, 225, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 251, 252, 254, 256, 258, 260, 261, 262, 264, 266, 268, 270, 272,274, 275, 276, 278, 280, 282, 283, 284, 285, 286, 288, 289, 290, 292,294, 295, 296, 297, 298, 299, 300, 302, 303, 305, 306, 308, 309, 310,311, 312, 314, 316, 317, 318, 320, 321, 323, 325, 326, 327, 328, 329,331, 333, 335, 336, 337, 338, 339, 340, 342, 344, 346, 348, 350, 351,353, 355, 357, 359, 360, 361, 362, 363, 364, 365, 366, 368, 370, 372,373, 374, 375, 376, 377, 379, 380, 381, 383, 384, 386, 388, 390, 392,393, 394, 396, 398, 400, 402, 404, 406, 407, 408, 410, 412, 413, 414,416, 418, 419, 420, 422, 423, 424, 426, 427, 429, 430, 431, 433, 434,436, 437, 439, 441, 442, 444, 446, 448, 449, 450, 451, 452, 454, 456,458, 459, 460, 462, 463, 465, 466, 468, 470, 472, 473, 474, 476, 478,479, 480, 481, 483, 485, 486, 488, 490, 492, 493, 495, 497, 498, 500,501, 503, 504, 506, 508, 509, 510, 512, 514, 515, 516, 517, 519, 521,522, 523, 525, 527, 529, 531, 533, 534, 535, 537, 539, 541, 543, 545,547, 549, 551, 552, 554, 556, 557, 559, 562, 564, 565, 567, 568, 570,572, 573, 574, 575, 576, 578, 580, 582, 584, 586, 588, 589, 590, 592,594, 596, 598, 599, 601, 602, 603, 605, 607, 608, 609, 611, 613, 615,617, 619, 621, 622, 624, 625, 627, 629, 630, 632, 634, 636, 638, 641,643, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 656, 658, 660,662, 664, 666, 668, 670, 672, 674, 676, 677, 679, 680, 682, 684, 686,687, 688, 689, 690, 691, 692, 694, 695, 697, 699, 701, 702, 704, 706,708, 710, 712, 713, 715, 716, 718, 720, 721, 723, 724, 726, 727, 729,730, 732, 733, 735, 736, 737, 739, 740, 742, 744, 746, 747, 748, 749,750, 751, 753, 755, 757, 758, 760, 761, 763, 764, 765, 766, 767, 768,769, 771, 772, 774, 775, 776, 777, 778, 780, 781, 782, 783, 785, 786,788, 789, 791, 793, 795, 796, 798, 799, 800, 801, 802, 803, 805, 807,808, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, and 823are provided in FIGS. 1-12 and in the Sequence Listing.

“Percent sequence identity” refers to the degree of sequence identitybetween any given reference sequence, e.g., SEQ ID NO: 1, and acandidate biomass composition-modulating sequence. A candidate sequencetypically has a length that is from 80 percent to 200 percent of thelength of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97,99, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, or 200percent of the length of the reference sequence. A percent identity forany candidate nucleic acid or polypeptide relative to a referencenucleic acid or polypeptide can be determined as follows. A referencesequence (e.g., a nucleic acid sequence or an amino acid sequence) isaligned to one or more candidate sequences using the computer programClustalW (version 1.83, default parameters), which allows alignments ofnucleic acid or polypeptide sequences to be carried out across theirentire length (global alignment). Chenna et al., Nucleic Acids Res.,31(13):3497-500 (2003).

ClustalW calculates the best match between a reference and one or morecandidate sequences, and aligns them so that identities, similaritiesand differences can be determined. Gaps of one or more residues can beinserted into a reference sequence, a candidate sequence, or both, tomaximize sequence alignments. For fast pairwise alignment of nucleicacid sequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For multiple alignment of nucleic acid sequences,the following parameters are used: gap opening penalty: 10.0; gapextension penalty: 5.0; and weight transitions: yes. For fast pairwisealignment of protein sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of proteinsequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps:on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, andLys; residue-specific gap penalties: on. The ClustalW output is asequence alignment that reflects the relationship between sequences.ClustalW can be run, for example, at the Baylor College of MedicineSearch Launcher site on the World Wide Web(searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at theEuropean Bioinformatics Institute site on the World Wide Web(ebi.ac.uk/clustalw).

To determine percent identity of a candidate nucleic acid or amino acidsequence to a reference sequence, the sequences are aligned usingClustalW, the number of identical matches in the alignment is divided bythe length of the reference sequence, and the result is multiplied by100. It is noted that the percent identity value can be rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded upto 78.2.

In some cases, a biomass composition-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 483 Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 483 are provided in FIG. 1and in the Sequence Listing.

In some cases, a biomass composition-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 562 Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 562 are provided in FIG. 2and in the Sequence Listing.

In some cases, a biomass composition-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 246 Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 246 are provided in FIG. 3and in the Sequence Listing.

In some cases, a biomass composition-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 111 Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 111 are provided in FIG. 4and in the Sequence Listing.

In some cases, a biomass composition-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 348 Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 348 are provided in FIG. 5and in the Sequence Listing.

In some cases, a biomass composition-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 774 Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 774 are provided in FIG. 6and in the Sequence Listing.

In some cases, a biomass composition-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 416 Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 416 are provided in FIG. 7and in the Sequence Listing.

In some cases, a biomass composition-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 2. Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 2 are provided in FIG. 8 andin the Sequence Listing.

In some cases, a biomass composition-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 157 Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 157 are provided in FIG. 9and in the Sequence Listing.

In some cases, a biomass composition-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 280 Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 280 are provided in FIG. 10and in the Sequence Listing.

In some cases, a biomass composition-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 641 Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 641 are provided in FIG. 11and in the Sequence Listing.

In some cases, a biomass composition-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 26. Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 26 are provided in FIG. 12and in the Sequence Listing.

E. Other Sequences

It should be appreciated that a biomass composition-modulatingpolypeptide can include additional amino acids that are not involved inbiomass modulation, and thus such a polypeptide can be longer than wouldotherwise be the case. For example, a biomass composition-modulatingpolypeptide can include a purification tag, a chloroplast transitpeptide, a mitochondrial transit peptide, an amyloplast peptide, or aleader sequence added to the amino or carboxy terminus. In someembodiments, a biomass composition-modulating polypeptide includes anamino acid sequence that functions as a reporter, e.g., a greenfluorescent protein or yellow fluorescent protein.

III. NUCLEIC ACIDS

Nucleic acids described herein include nucleic acids that are effectiveto modulate biomass composition when transcribed in a plant or plantcell. Such nucleic acids include, without limitation, those that encodea biomass composition-modulating polypeptide and those that can be usedto inhibit expression of a biomass composition-modulating polypeptidevia a nucleic acid based method.

A. Nucleic Acids Encoding Biomass Composition-Modulating Polypeptides

Nucleic acids encoding biomass composition-modulating polypeptides aredescribed herein. Examples of such nucleic acids include SEQ ID NOs: 1,3, 5, 9, 11, 13, 16, 23, 25, 27, 31, 33, 35, 38, 40, 42, 44, 46, 48, 51,54, 56, 58, 60, 62, 64, 67, 69, 74, 76, 80, 83, 85, 87, 89, 91, 93, 95,98, 103, 106, 108, 110, 112, 114, 116, 119, 121, 123, 125, 127, 129,131, 134, 137, 140, 142, 144, 146, 150, 154, 156, 159, 166, 169, 174,176, 178, 180, 183, 186, 188, 191, 193, 195, 198, 200, 203, 206, 209,212, 214, 217, 219, 224, 227, 229, 231, 233, 235, 237, 239, 241, 243,245, 247, 249, 253, 255, 257, 259, 263, 265, 267, 269, 271, 273, 277,279, 281, 287, 291, 293, 301, 304, 307, 313, 315, 319, 322, 324, 330,332, 334, 341, 343, 345, 347, 349, 352, 354, 356, 358, 367, 369, 371,378, 382, 385, 387, 389, 391, 395, 397, 399, 401, 403, 405, 409, 411,415, 417, 421, 425, 428, 432, 435, 438, 440, 443, 445, 447, 453, 455,457, 461, 464, 467, 469, 471, 475, 477, 482, 484, 487, 489, 491, 494,496, 499, 502, 505, 507, 511, 513, 518, 520, 524, 526, 528, 530, 532,536, 538, 540, 542, 544, 546, 548, 550, 553, 555, 558, 560, 561, 563,566, 569, 571, 577, 579, 581, 583, 585, 587, 591, 593, 595, 597, 600,604, 606, 610, 612, 614, 616, 618, 620, 623, 626, 628, 631, 633, 635,637, 639, 640, 642, 644, 655, 657, 659, 661, 663, 665, 667, 669, 671,673, 675, 678, 681, 683, 685, 693, 696, 698, 700, 703, 705, 707, 709,711, 714, 717, 719, 722, 725, 728, 731, 734, 738, 741, 743, 745, 752,754, 756, 759, 762, 770, 773, 779, 784, 787, 790, 792, 794, 797, 804,806, 809, and 822, as described in more detail below. A nucleic acidalso can be a fragment that is at least 40% (e.g., at least 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, or 99%) of the length of the full-lengthnucleic acid set forth in SEQ ID NOs: 1, 3, 5, 9, 11, 13, 16, 23, 25,27, 31, 33, 35, 38, 40, 42, 44, 46, 48, 51, 54, 56, 58, 60, 62, 64, 67,69, 74, 76, 80, 83, 85, 87, 89, 91, 93, 95, 98, 103, 106, 108, 110, 112,114, 116, 119, 121, 123, 125, 127, 129, 131, 134, 137, 140, 142, 144,146, 150, 154, 156, 159, 166, 169, 174, 176, 178, 180, 183, 186, 188,191, 193, 195, 198, 200, 203, 206, 209, 212, 214, 217, 219, 224, 227,229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 253, 255, 257,259, 263, 265, 267, 269, 271, 273, 277, 279, 281, 287, 291, 293, 301,304, 307, 313, 315, 319, 322, 324, 330, 332, 334, 341, 343, 345, 347,349, 352, 354, 356, 358, 367, 369, 371, 378, 382, 385, 387, 389, 391,395, 397, 399, 401, 403, 405, 409, 411, 415, 417, 421, 425, 428, 432,435, 438, 440, 443, 445, 447, 453, 455, 457, 461, 464, 467, 469, 471,475, 477, 482, 484, 487, 489, 491, 494, 496, 499, 502, 505, 507, 511,513, 518, 520, 524, 526, 528, 530, 532, 536, 538, 540, 542, 544, 546,548, 550, 553, 555, 558, 560, 561, 563, 566, 569, 571, 577, 579, 581,583, 585, 587, 591, 593, 595, 597, 600, 604, 606, 610, 612, 614, 616,618, 620, 623, 626, 628, 631, 633, 635, 637, 639, 640, 642, 644, 655,657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 678, 681, 683, 685,693, 696, 698, 700, 703, 705, 707, 709, 711, 714, 717, 719, 722, 725,728, 731, 734, 738, 741, 743, 745, 752, 754, 756, 759, 762, 770, 773,779, 784, 787, 790, 792, 794, 797, 804, 806, 809, and 822.

A biomass composition-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO: 482. Alternatively, abiomass composition-modulating nucleic acid can be a variant of thenucleic acid having the nucleotide sequence set forth in SEQ ID NO: 482.For example, a biomass composition-modulating nucleic acid can have anucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequenceset forth in SEQ ID NO: 482.

A biomass composition-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO: 561. Alternatively, abiomass composition-modulating nucleic acid can be a variant of thenucleic acid having the nucleotide sequence set forth in SEQ ID NO: 561.For example, a biomass composition-modulating nucleic acid can have anucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequenceset forth in SEQ ID NO: 561.

A biomass composition-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO: 245. Alternatively, abiomass composition-modulating nucleic acid can be a variant of thenucleic acid having the nucleotide sequence set forth in SEQ ID NO: 245.For example, a biomass composition-modulating nucleic acid can have anucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequenceset forth in SEQ ID NO: 245.

A biomass composition-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO: 110. Alternatively, abiomass composition-modulating nucleic acid can be a variant of thenucleic acid having the nucleotide sequence set forth in SEQ ID NO: 110.For example, a biomass composition-modulating nucleic acid can have anucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequenceset forth in SEQ ID NO: 110.

A biomass composition-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO: 347. Alternatively, abiomass composition-modulating nucleic acid can be a variant of thenucleic acid having the nucleotide sequence set forth in SEQ ID NO: 347.For example, a biomass composition-modulating nucleic acid can have anucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequenceset forth in SEQ ID NO: 347.

A biomass composition-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO: 773. Alternatively, abiomass composition-modulating nucleic acid can be a variant of thenucleic acid having the nucleotide sequence set forth in SEQ ID NO: 773.For example, a biomass composition-modulating nucleic acid can have anucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequenceset forth in SEQ ID NO: 773.

A biomass composition-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO: 415. Alternatively, abiomass composition-modulating nucleic acid can be a variant of thenucleic acid having the nucleotide sequence set forth in SEQ ID NO: 415.For example, a biomass composition-modulating nucleic acid can have anucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequenceset forth in SEQ ID NO: 415.

A biomass composition-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO: 1 or a fragment of thenucleotide sequence set forth in SEQ ID NO: 1. For example, a deletioncan be made at nucleotide position 657 of SEQ ID NO: 1 such that atruncated protein is encoded (e.g., a truncated protein having about 142residues). Alternatively, a biomass composition-modulating nucleic acidcan be a variant of the nucleic acid having the nucleotide sequence setforth in SEQ ID NO: 1. For example, a biomass composition-modulatingnucleic acid can have a nucleotide sequence with at least 80% sequenceidentity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity,to the nucleotide sequence set forth in SEQ ID NO: 1. Such variantbiomass composition-modulating nucleotide sequences can have a deletionat the nucleotide position corresponding to position 657 of SEQ ID NO: 1such that a truncated protein is encoded.

A biomass composition-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO: 156. Alternatively, abiomass composition-modulating nucleic acid can be a variant of thenucleic acid having the nucleotide sequence set forth in SEQ ID NO: 156.For example, a biomass composition-modulating nucleic acid can have anucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequenceset forth in SEQ ID NO: 156.

A biomass composition-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO: 279. Alternatively, abiomass composition-modulating nucleic acid can be a variant of thenucleic acid having the nucleotide sequence set forth in SEQ ID NO: 279.For example, a biomass composition-modulating nucleic acid can have anucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequenceset forth in SEQ ID NO: 279.

A biomass composition-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO: 640. Alternatively, abiomass composition-modulating nucleic acid can be a variant of thenucleic acid having the nucleotide sequence set forth in SEQ ID NO: 640.For example, a biomass composition-modulating nucleic acid can have anucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequenceset forth in SEQ ID NO: 640.

A biomass composition-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO: 25. Alternatively, a biomasscomposition-modulating nucleic acid can be a variant of the nucleic acidhaving the nucleotide sequence set forth in SEQ ID NO: 25. For example,a biomass composition-modulating nucleic acid can have a nucleotidesequence with at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%,97%, 98%, or 99% sequence identity, to the nucleotide sequence set forthin SEQ ID NO: 25.

Isolated nucleic acid molecules can be produced by standard techniques.For example, polymerase chain reaction (PCR) techniques can be used toobtain an isolated nucleic acid containing a nucleotide sequencedescribed herein. PCR can be used to amplify specific sequences from DNAas well as RNA, including sequences from total genomic DNA or totalcellular RNA. Various PCR methods are described, for example, in PCRPrimer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold SpringHarbor Laboratory Press, 1995. Generally, sequence information from theends of the region of interest or beyond is employed to designoligonucleotide primers that are identical or similar in sequence toopposite strands of the template to be amplified. Various PCR strategiesalso are available by which site-specific nucleotide sequencemodifications can be introduced into a template nucleic acid. Isolatednucleic acids also can be chemically synthesized, either as a singlenucleic acid molecule (e.g., using automated DNA synthesis in the 3′ to5′ direction using phosphoramidite technology) or as a series ofoligonucleotides. For example, one or more pairs of longoligonucleotides (e.g., >100 nucleotides) can be synthesized thatcontain the desired sequence, with each pair containing a short segmentof complementarity (e.g., about 15 nucleotides) such that a duplex isformed when the oligonucleotide pair is annealed. DNA polymerase is usedto extend the oligonucleotides, resulting in a single, double-strandednucleic acid molecule per oligonucleotide pair, which then can beligated into a vector. Isolated nucleic acids of the invention also canbe obtained by mutagenesis of, e.g., a naturally occurring DNA.

B. Use of Nucleic Acids to Modulate Expression of Polypeptides

i. Expression of a Biomass Composition-Modulating Polypeptide

A nucleic acid encoding one of the biomass composition-modulatingpolypeptides described herein can be used to express the polypeptide ina plant species of interest, typically by transforming a plant cell witha nucleic acid having the coding sequence for the polypeptide operablylinked in sense orientation to one or more regulatory regions. It willbe appreciated that because of the degeneracy of the genetic code, anumber of nucleic acids can encode a particular biomasscomposition-modulating polypeptide; i.e., for many amino acids, there ismore than one nucleotide triplet that serves as the codon for the aminoacid. Thus, codons in the coding sequence for a given biomasscomposition-modulating polypeptide can be modified such that optimalexpression in a particular plant species is obtained, using appropriatecodon bias tables for that species.

In some cases, expression of a biomass composition-modulatingpolypeptide inhibits one or more functions of an endogenous polypeptide.For example, a nucleic acid that encodes a dominant negative polypeptidecan be used to inhibit protein function. A dominant negative polypeptidetypically is mutated or truncated relative to an endogenous wild typepolypeptide, and its presence in a cell inhibits one or more functionsof the wild type polypeptide in that cell, i.e., the dominant negativepolypeptide is genetically dominant and confers a loss of function. Themechanism by which a dominant negative polypeptide confers such aphenotype can vary but often involves a protein-protein interaction or aprotein-DNA interaction. For example, a dominant negative polypeptidecan be an enzyme that is truncated relative to a native wild typeenzyme, such that the truncated polypeptide retains domains involved inbinding a first protein but lacks domains involved in binding a secondprotein. The truncated polypeptide is thus unable to properly modulatethe activity of the second protein. See, e.g., US 2007/0056058. Asanother example, a point mutation that results in a non-conservativeamino acid substitution in a catalytic domain can result in a dominantnegative polypeptide. See, e.g., US 2005/032221. As another example, adominant negative polypeptide can be a transcription factor that istruncated relative to a native wild type transcription factor, such thatthe truncated polypeptide retains the DNA binding domain(s) but lacksthe activation domain(s). Such a truncated polypeptide can inhibit thewild type transcription factor from binding DNA, thereby inhibitingtranscription activation.

ii. Inhibition of Expression of a Biomass Composition-ModulatingPolypeptide

Polynucleotides and recombinant constructs described herein can be usedto inhibit expression of a biomass composition-modulating polypeptide ina plant species of interest. See, e.g., Matzke and Birchler, NatureReviews Genetics 6:24-35 (2005); Akashi et al., Nature Reviews Mol. CellBiology 6:413-422 (2005); Mittal, Nature Reviews Genetics 5:355-365(2004); and Nature Reviews RNA interference collection, October 2005 onthe World Wide Web at nature.com/reviews/focus/mai. A number of nucleicacid based methods, including antisense RNA, ribozyme directed RNAcleavage, post-transcriptional gene silencing (PTGS), e.g., RNAinterference (RNAi), and transcriptional gene silencing (TGS) are knownto inhibit gene expression in plants. Suitable polynucleotides includefull-length nucleic acids encoding biomass composition-modulatingpolypeptides or fragments of such full-length nucleic acids. In someembodiments, a complement of the full-length nucleic acid or a fragmentthereof can be used. Typically, a fragment is at least 10 nucleotides,e.g., at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 30, 35, 40, 50, 80, 100, 200, 500 nucleotides or more.Generally, higher homology can be used to compensate for the use of ashorter sequence.

Antisense technology is one well-known method. In this method, a nucleicacid of a gene to be repressed is cloned and operably linked to aregulatory region and a transcription termination sequence so that theantisense strand of RNA is transcribed. The recombinant construct isthen transformed into plants, as described herein, and the antisensestrand of RNA is produced. The nucleic acid need not be the entiresequence of the gene to be repressed, but typically will besubstantially complementary to at least a portion of the sense strand ofthe gene to be repressed.

In another method, a nucleic acid can be transcribed into a ribozyme, orcatalytic RNA, that affects expression of an mRNA. See, U.S. Pat. No.6,423,885. Ribozymes can be designed to specifically pair with virtuallyany target RNA and cleave the phosphodiester backbone at a specificlocation, thereby functionally inactivating the target RNA. Heterologousnucleic acids can encode ribozymes designed to cleave particular mRNAtranscripts, thus preventing expression of a polypeptide. Hammerheadribozymes are useful for destroying particular mRNAs, although variousribozymes that cleave mRNA at site-specific recognition sequences can beused. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target RNA contains a 5′-UG-3′nucleotide sequence. The construction and production of hammerheadribozymes is known in the art. See, for example, U.S. Pat. No. 5,254,678and WO 02/46449 and references cited therein. Hammerhead ribozymesequences can be embedded in a stable RNA such as a transfer RNA (tRNA)to increase cleavage efficiency in vivo. Perriman et al., Proc. Natl.Acad. Sci. USA, 92(13):6175-6179 (1995); de Feyter and Gaudron, Methodsin Molecular Biology, Vol. 74, Chapter 43, “Expressing Ribozymes inPlants”, Edited by Turner, P. C., Humana Press Inc., Totowa, N.J. RNAendoribonucleases which have been described, such as the one that occursnaturally in Tetrahymena thermophila, can be useful. See, for example,U.S. Pat. Nos. 4,987,071 and 6,423,885.

PTGS, e.g., RNAi, can also be used to inhibit the expression of a gene.For example, a construct can be prepared that includes a sequence thatis transcribed into an RNA that can anneal to itself, e.g., a doublestranded RNA having a stem-loop structure. In some embodiments, onestrand of the stem portion of a double stranded RNA comprises a sequencethat is similar or identical to the sense coding sequence or a fragmentthereof of a biomass composition-modulating polypeptide, and that isfrom about 10 nucleotides to about 2,500 nucleotides in length. Thelength of the sequence that is similar or identical to the sense codingsequence can be from 10 nucleotides to 500 nucleotides, from 15nucleotides to 300 nucleotides, from 20 nucleotides to 100 nucleotides,or from 25 nucleotides to 100 nucleotides. The other strand of the stemportion of a double stranded RNA comprises a sequence that is similar oridentical to the antisense strand or a fragment thereof of the codingsequence of the biomass composition-modulating polypeptide, and can havea length that is shorter, the same as, or longer than the correspondinglength of the sense sequence. In some cases, one strand of the stemportion of a double stranded RNA comprises a sequence that is similar oridentical to the 3′ or 5′ untranslated region, or a fragment thereof, ofan mRNA encoding a biomass composition-modulating polypeptide, and theother strand of the stem portion of the double stranded RNA comprises asequence that is similar or identical to the sequence that iscomplementary to the 3′ or 5′ untranslated region, respectively, or afragment thereof, of the mRNA encoding the biomasscomposition-modulating polypeptide. In other embodiments, one strand ofthe stem portion of a double stranded RNA comprises a sequence that issimilar or identical to the sequence of an intron, or a fragmentthereof, in the pre-mRNA encoding a biomass composition-modulatingpolypeptide, and the other strand of the stem portion comprises asequence that is similar or identical to the sequence that iscomplementary to the sequence of the intron, or a fragment thereof, inthe pre-mRNA.

The loop portion of a double stranded RNA can be from 3 nucleotides to5,000 nucleotides, e.g., from 3 nucleotides to 25 nucleotides, from 15nucleotides to 1,000 nucleotides, from 20 nucleotides to 500nucleotides, or from 25 nucleotides to 200 nucleotides. The loop portionof the RNA can include an intron or a fragment thereof. A doublestranded RNA can have zero, one, two, three, four, five, six, seven,eight, nine, ten, or more stem-loop structures.

A construct including a sequence that is operably linked to a regulatoryregion and a transcription termination sequence, and that is transcribedinto an RNA that can form a double stranded RNA, is transformed intoplants as described herein. Methods for using RNAi to inhibit theexpression of a gene are known to those of skill in the art. See, e.g.,U.S. Pat. Nos. 5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753,139;and 6,777,588. See also WO 97/01952; WO 98/53083; WO 99/32619; WO98/36083; and U.S. Patent Publications 20030175965, 20030175783,20040214330, and 20030180945.

Constructs containing regulatory regions operably linked to nucleic acidmolecules in sense orientation can also be used to inhibit theexpression of a gene. The transcription product can be similar oridentical to the sense coding sequence, or a fragment thereof, of abiomass composition-modulating polypeptide. The transcription productalso can be unpolyadenylated, lack a 5′ cap structure, or contain anunspliceable intron. Methods of inhibiting gene expression using afull-length cDNA as well as a partial cDNA sequence are known in theart. See, e.g., U.S. Pat. No. 5,231,020.

In some embodiments, a construct containing a nucleic acid having atleast one strand that is a template for both sense and antisensesequences that are complementary to each other is used to inhibit theexpression of a gene. The sense and antisense sequences can be part of alarger nucleic acid molecule or can be part of separate nucleic acidmolecules having sequences that are not complementary. The sense orantisense sequence can be a sequence that is identical or complementaryto the sequence of an mRNA, the 3′ or 5′ untranslated region of an mRNA,or an intron in a pre-mRNA encoding a biomass composition-modulatingpolypeptide, or a fragment of such sequences. In some embodiments, thesense or antisense sequence is identical or complementary to a sequenceof the regulatory region that drives transcription of the gene encodinga biomass composition-modulating polypeptide. In each case, the sensesequence is the sequence that is complementary to the antisensesequence.

The sense and antisense sequences can be a length greater than about 10nucleotides (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, or more nucleotides). For example, an antisensesequence can be 21 or 22 nucleotides in length. Typically, the sense andantisense sequences range in length from about 15 nucleotides to about30 nucleotides, e.g., from about 18 nucleotides to about 28 nucleotides,or from about 21 nucleotides to about 25 nucleotides.

In some embodiments, an antisense sequence is a sequence complementaryto an mRNA sequence, or a fragment thereof, encoding a biomasscomposition-modulating polypeptide described herein. The sense sequencecomplementary to the antisense sequence can be a sequence present withinthe mRNA of the biomass composition-modulating polypeptide. Typically,sense and antisense sequences are designed to correspond to a 15-30nucleotide sequence of a target mRNA such that the level of that targetmRNA is reduced.

In some embodiments, a construct containing a nucleic acid having atleast one strand that is a template for more than one sense sequence(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sense sequences) can be usedto inhibit the expression of a gene. Likewise, a construct containing anucleic acid having at least one strand that is a template for more thanone antisense sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreantisense sequences) can be used to inhibit the expression of a gene.For example, a construct can contain a nucleic acid having at least onestrand that is a template for two sense sequences and two antisensesequences. The multiple sense sequences can be identical or different,and the multiple antisense sequences can be identical or different. Forexample, a construct can have a nucleic acid having one strand that is atemplate for two identical sense sequences and two identical antisensesequences that are complementary to the two identical sense sequences.Alternatively, an isolated nucleic acid can have one strand that is atemplate for (1) two identical sense sequences 20 nucleotides in length,(2) one antisense sequence that is complementary to the two identicalsense sequences 20 nucleotides in length, (3) a sense sequence 30nucleotides in length, and (4) three identical antisense sequences thatare complementary to the sense sequence 30 nucleotides in length. Theconstructs provided herein can be designed to have a suitablearrangement of sense and antisense sequences. For example, two identicalsense sequences can be followed by two identical antisense sequences orcan be positioned between two identical antisense sequences.

A nucleic acid having at least one strand that is a template for one ormore sense and/or antisense sequences can be operably linked to aregulatory region to drive transcription of an RNA molecule containingthe sense and/or antisense sequence(s). In addition, such a nucleic acidcan be operably linked to a transcription terminator sequence, such asthe terminator of the nopaline synthase (nos) gene. In some cases, tworegulatory regions can direct transcription of two transcripts: one fromthe top strand, and one from the bottom strand. See, for example, Yan etal., Plant Physiol., 141:1508-1518 (2006). The two regulatory regionscan be the same or different. The two transcripts can formdouble-stranded RNA molecules that induce degradation of the target RNA.In some cases, a nucleic acid can be positioned within a T-DNA orplant-derived transfer DNA (P-DNA) such that the left and right T-DNAborder sequences or the left and right border-like sequences of theP-DNA flank, or are on either side of, the nucleic acid. See, US2006/0265788. The nucleic acid sequence between the two regulatoryregions can be from about 15 to about 300 nucleotides in length. In someembodiments, the nucleic acid sequence between the two regulatoryregions is from about 15 to about 200 nucleotides in length, from about15 to about 100 nucleotides in length, from about 15 to about 50nucleotides in length, from about 18 to about 50 nucleotides in length,from about 18 to about 40 nucleotides in length, from about 18 to about30 nucleotides in length, or from about 18 to about 25 nucleotides inlength.

In some nucleic-acid based methods for inhibition of gene expression inplants, a suitable nucleic acid can be a nucleic acid analog. Nucleicacid analogs can be modified at the base moiety, sugar moiety, orphosphate backbone to improve, for example, stability, hybridization, orsolubility of the nucleic acid. Modifications at the base moiety includedeoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine and5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugarmoiety include modification of the 2′ hydroxyl of the ribose sugar toform 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphatebackbone can be modified to produce morpholino nucleic acids, in whicheach base moiety is linked to a six-membered morpholino ring, or peptidenucleic acids, in which the deoxyphosphate backbone is replaced by apseudopeptide backbone and the four bases are retained. See, forexample, Summerton and Weller, Antisense Nucleic Acid Drug Dev.,7:187-195 (1997); Hyrup et al., Bioorgan. Med. Chem., 4:5-23 (1996). Inaddition, the deoxyphosphate backbone can be replaced with, for example,a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite,or an alkyl phosphotriester backbone.

C. Constructs/Vectors

Recombinant constructs provided herein can be used to transform plantsor plant cells in order to modulate biomass levels. A recombinantnucleic acid construct can comprise a nucleic acid encoding a biomasscomposition-modulating polypeptide as described herein, operably linkedto a regulatory region suitable for expressing the biomasscomposition-modulating polypeptide in the plant or cell. Thus, a nucleicacid can comprise a coding sequence that encodes a biomasscomposition-modulating polypeptides as set forth in SEQ ID NOs: 2, 4, 6,7, 8, 10, 12, 14, 15, 17, 18, 19, 20, 21, 22, 24, 26, 28, 29, 30, 32,34, 36, 37, 39, 41, 43, 45, 47, 49, 50, 52, 53, 55, 57, 59, 61, 63, 65,66, 68, 70, 71, 72, 73, 75, 77, 78, 79, 81, 82, 84, 86, 88, 90, 92, 94,96, 97, 99, 100, 101, 102, 104, 105, 107, 109, 111, 113, 115, 117, 118,120, 122, 124, 126, 128, 130, 132, 133, 135, 136, 138, 139, 141, 143,145, 147, 148, 149, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163,164, 165, 167, 168, 170, 171, 172, 173, 175, 177, 179, 181, 182, 184,185, 187, 189, 190, 192, 194, 196, 197, 199, 201, 202, 204, 205, 207,208, 210, 211, 213, 215, 216, 218, 220, 221, 222, 223, 225, 226, 228,230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 251, 252, 254,256, 258, 260, 261, 262, 264, 266, 268, 270, 272, 274, 275, 276, 278,280, 282, 283, 284, 285, 286, 288, 289, 290, 292, 294, 295, 296, 297,298, 299, 300, 302, 303, 305, 306, 308, 309, 310, 311, 312, 314, 316,317, 318, 320, 321, 323, 325, 326, 327, 328, 329, 331, 333, 335, 336,337, 338, 339, 340, 342, 344, 346, 348, 350, 351, 353, 355, 357, 359,360, 361, 362, 363, 364, 365, 366, 368, 370, 372, 373, 374, 375, 376,377, 379, 380, 381, 383, 384, 386, 388, 390, 392, 393, 394, 396, 398,400, 402, 404, 406, 407, 408, 410, 412, 413, 414, 416, 418, 419, 420,422, 423, 424, 426, 427, 429, 430, 431, 433, 434, 436, 437, 439, 441,442, 444, 446, 448, 449, 450, 451, 452, 454, 456, 458, 459, 460, 462,463, 465, 466, 468, 470, 472, 473, 474, 476, 478, 479, 480, 481, 483,485, 486, 488, 490, 492, 493, 495, 497, 498, 500, 501, 503, 504, 506,508, 509, 510, 512, 514, 515, 516, 517, 519, 521, 522, 523, 525, 527,529, 531, 533, 534, 535, 537, 539, 541, 543, 545, 547, 549, 551, 552,554, 556, 557, 559, 562, 564, 565, 567, 568, 570, 572, 573, 574, 575,576, 578, 580, 582, 584, 586, 588, 589, 590, 592, 594, 596, 598, 599,601, 602, 603, 605, 607, 608, 609, 611, 613, 615, 617, 619, 621, 622,624, 625, 627, 629, 630, 632, 634, 636, 638, 641, 643, 645, 646, 647,648, 649, 650, 651, 652, 653, 654, 656, 658, 660, 662, 664, 666, 668,670, 672, 674, 676, 677, 679, 680, 682, 684, 686, 687, 688, 689, 690,691, 692, 694, 695, 697, 699, 701, 702, 704, 706, 708, 710, 712, 713,715, 716, 718, 720, 721, 723, 724, 726, 727, 729, 730, 732, 733, 735,736, 737, 739, 740, 742, 744, 746, 747, 748, 749, 750, 751, 753, 755,757, 758, 760, 761, 763, 764, 765, 766, 767, 768, 769, 771, 772, 774,775, 776, 777, 778, 780, 781, 782, 783, 785, 786, 788, 789, 791, 793,795, 796, 798, 799, 800, 801, 802, 803, 805, 807, 808, 810, 811, 812,813, 814, 815, 816, 817, 818, 819, 820, 821, and 823. Examples ofnucleic acids encoding biomass composition-modulating polypeptides areset forth in SEQ ID NOs: 1, 3, 5, 9, 11, 13, 16, 23, 25, 27, 31, 33, 35,38, 40, 42, 44, 46, 48, 51, 54, 56, 58, 60, 62, 64, 67, 69, 74, 76, 80,83, 85, 87, 89, 91, 93, 95, 98, 103, 106, 108, 110, 112, 114, 116, 119,121, 123, 125, 127, 129, 131, 134, 137, 140, 142, 144, 146, 150, 154,156, 159, 166, 169, 174, 176, 178, 180, 183, 186, 188, 191, 193, 195,198, 200, 203, 206, 209, 212, 214, 217, 219, 224, 227, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 253, 255, 257, 259, 263, 265,267, 269, 271, 273, 277, 279, 281, 287, 291, 293, 301, 304, 307, 313,315, 319, 322, 324, 330, 332, 334, 341, 343, 345, 347, 349, 352, 354,356, 358, 367, 369, 371, 378, 382, 385, 387, 389, 391, 395, 397, 399,401, 403, 405, 409, 411, 415, 417, 421, 425, 428, 432, 435, 438, 440,443, 445, 447, 453, 455, 457, 461, 464, 467, 469, 471, 475, 477, 482,484, 487, 489, 491, 494, 496, 499, 502, 505, 507, 511, 513, 518, 520,524, 526, 528, 530, 532, 536, 538, 540, 542, 544, 546, 548, 550, 553,555, 558, 560, 561, 563, 566, 569, 571, 577, 579, 581, 583, 585, 587,591, 593, 595, 597, 600, 604, 606, 610, 612, 614, 616, 618, 620, 623,626, 628, 631, 633, 635, 637, 639, 640, 642, 644, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 678, 681, 683, 685, 693, 696, 698,700, 703, 705, 707, 709, 711, 714, 717, 719, 722, 725, 728, 731, 734,738, 741, 743, 745, 752, 754, 756, 759, 762, 770, 773, 779, 784, 787,790, 792, 794, 797, 804, 806, 809, and 822, or in the Sequence Listing.The biomass composition-modulating polypeptide encoded by a recombinantnucleic acid can be a native biomass composition-modulating polypeptide,or can be heterologous to the cell. In some cases, the recombinantconstruct contains a nucleic acid that inhibits expression of a biomasscomposition-modulating polypeptide, operably linked to a regulatoryregion. Examples of suitable regulatory regions are described in thesection entitled “Regulatory Regions.”

Vectors containing recombinant nucleic acid constructs such as thosedescribed herein also are provided. Suitable vector backbones include,for example, those routinely used in the art such as plasmids, viruses,artificial chromosomes, BACs, YACs, or PACs. Suitable expression vectorsinclude, without limitation, plasmids and viral vectors derived from,for example, bacteriophage, baculoviruses, and retroviruses. Numerousvectors and expression systems are commercially available from suchcorporations as Novagen® (Madison, Wis.), Clontech® (Palo Alto, Calif.),Stratagene® (La Jolla, Calif.), and Invitrogen/Life Technologies®(Carlsbad, Calif.).

The vectors provided herein also can include, for example, origins ofreplication, scaffold attachment regions (SARs), and/or markers. Amarker gene can confer a selectable phenotype on a plant cell. Forexample, a marker can confer biocide resistance, such as resistance toan antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin), or anherbicide (e.g., glyphosate, chlorsulfuron or phosphinothricin). Inaddition, an expression vector can include a tag sequence designed tofacilitate manipulation or detection (e.g., purification orlocalization) of the expressed polypeptide. Tag sequences, such asluciferase, β-glucuronidase (GUS), green fluorescent protein (GFP),glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, orFlag™ tag (Kodak, New Haven, Conn.) sequences typically are expressed asa fusion with the encoded polypeptide. Such tags can be insertedanywhere within the polypeptide, including at either the carboxyl oramino terminus.

D. Regulatory Regions

The choice of regulatory regions to be included in a recombinantconstruct depends upon several factors, including, but not limited to,efficiency, selectability, inducibility, desired expression level, andcell- or tissue-preferential expression. It is a routine matter for oneof skill in the art to modulate the expression of a coding sequence byappropriately selecting and positioning regulatory regions relative tothe coding sequence. Transcription of a nucleic acid can be modulated ina similar manner.

Some suitable regulatory regions initiate transcription only, orpredominantly, in certain cell types. Methods for identifying andcharacterizing regulatory regions in plant genomic DNA are known,including, for example, those described in the following references:Jordano et al., Plant Cell, 1:855-866 (1989); Bustos et al., Plant Cell,1:839-854 (1989); Green et al., EMBO J., 7:4035-4044 (1988); Meier etal., Plant Cell, 3:309-316 (1991); and Zhang et al., Plant Physiology,110:1069-1079 (1996).

Examples of various classes of regulatory regions are described below.Some of the regulatory regions indicated below as well as additionalregulatory regions are described in more detail in U.S. PatentApplication Ser. Nos. 60/505,689; 60/518,075; 60/544,771; 60/558,869;60/583,691; 60/619,181; 60/637,140; 60/757,544; 60/776,307; 10/957,569;11/058,689; 11/172,703; 11/208,308; 11/274,890; 60/583,609; 60/612,891;11/097,589; 11/233,726; 11/408,791; 11/414,142; 10/950,321; 11/360,017;PCT/US05/011105; PCT/US05/23639; PCT/US05/034308; PCT/US05/034343; andPCT/US06/038236; PCT/US06/040572; PCT/US07/62762; PCT/US2009/032485; andPCT/US2009/038792.

For example, the sequences of regulatory regions p326, YP0144, YP0190,p13879, YP0050, p32449, 21876, YP0158, YP0214, YP0380, PT0848, PT0633,YP0128, YP0275, PT0660, PT0683, PT0758, PT0613, PT0672, PT0688, PT0837,YP0092, PT0676, PT0708, YP0396, YP0007, YP0111, YP0103, YP0028, YP0121,YP0008, YP0039, YP0115, YP0119, YP0120, YP0374, YP0101, YP0102, YP0110,YP0117, YP0137, YP0285, YP0212, YP0097, YP0107, YP0088, YP0143, YP0156,PT0650, PT0695, PT0723, PT0838, PT0879, PT0740, PT0535, PT0668, PT0886,PT0585, YP0381, YP0337, PT0710, YP0356, YP0385, YP0384, YP0286, YP0377,PD1367, PT0863, PT0829, PT0665, PT0678, YP0086, YP0188, YP0263, PT0743and YP0096 are set forth in the sequence listing of PCT/US06/040572; thesequence of regulatory region PT0625 is set forth in the sequencelisting of PCT/US05/034343; the sequences of regulatory regions PT0623,YP0388, YP0087, YP0093, YP0108, YP0022 and YP0080 are set forth in thesequence listing of U.S. patent application Ser. No. 11/172,703; thesequence of regulatory region PR0924 is set forth in the sequencelisting of PCT/US07/62762; and the sequences of regulatory regionsp530c10, pOsFIE2-2, pOsMEA, pOsYp102, and pOsYp285 are set forth in thesequence listing of PCT/US06/038236.

It will be appreciated that a regulatory region may meet criteria forone classification based on its activity in one plant species, and yetmeet criteria for a different classification based on its activity inanother plant species.

i. Broadly Expressing Promoters

A promoter can be said to be “broadly expressing” when it promotestranscription in many, but not necessarily all, plant tissues. Forexample, a broadly expressing promoter can promote transcription of anoperably linked sequence in one or more of the shoot, shoot tip (apex),and leaves, but weakly or not at all in tissues such as roots or stems.As another example, a broadly expressing promoter can promotetranscription of an operably linked sequence in one or more of the stem,shoot, shoot tip (apex), and leaves, but can promote transcriptionweakly or not at all in tissues such as reproductive tissues of flowersand developing seeds. Non-limiting examples of broadly expressingpromoters that can be included in the nucleic acid constructs providedherein include the p326, YP0144, YP0190, p13879, YP0050, p32449, 21876,YP0158, YP0214, YP0380, PT0848, and PT0633 promoters. Additionalexamples include the cauliflower mosaic virus (CaMV) 35S promoter, themannopine synthase (MAS) promoter, the 1′ or 2′ promoters derived fromT-DNA of Agrobacterium tumefaciens, the figwort mosaic virus 34Spromoter, actin promoters such as the rice actin promoter, and ubiquitinpromoters such as the maize ubiquitin-1 promoter. In some cases, theCaMV 35S promoter is excluded from the category of broadly expressingpromoters.

ii. Root Promoters

Root-active promoters confer transcription in root tissue, e.g., rootendodermis, root epidermis, or root vascular tissues. In someembodiments, root-active promoters are root-preferential promoters,i.e., confer transcription only or predominantly in root tissue.Root-preferential promoters include the YP0128, YP0275, PT0625, PT0660,PT0683, and PT0758 promoters. Other root-preferential promoters includethe PT0613, PT0672, PT0688, and PT0837 promoters, which drivetranscription primarily in root tissue and to a lesser extent in ovulesand/or seeds. Other examples of root-preferential promoters include theroot-specific subdomains of the CaMV 35S promoter (Lam et al., Proc.Natl. Acad. Sci. USA, 86:7890-7894 (1989)), root cell specific promotersreported by Conkling et al., Plant Physiol., 93:1203-1211 (1990), andthe tobacco RD2 promoter.

iii. Maturing Endosperm Promoters

In some embodiments, promoters that drive transcription in maturingendosperm can be useful. Transcription from a maturing endospermpromoter typically begins after fertilization and occurs primarily inendosperm tissue during seed development and is typically highest duringthe cellularization phase. Most suitable are promoters that are activepredominantly in maturing endosperm, although promoters that are alsoactive in other tissues can sometimes be used. Non-limiting examples ofmaturing endosperm promoters that can be included in the nucleic acidconstructs provided herein include the napin promoter, the Arcelin-5promoter, the phaseolin promoter (Bustos et al., Plant Cell,1(9):839-853 (1989)), the soybean trypsin inhibitor promoter (Riggs etal., Plant Cell, 1(6):609-621 (1989)), the ACP promoter (Baerson et al.,Plant Mol. Biol., 22(2):255-267 (1993)), the stearoyl-ACP desaturasepromoter (Slocombe et al., Plant Physiol., 104(4):167-176 (1994)), thesoybean α′ subunit of β-conglycinin promoter (Chen et al., Proc. Natl.Acad. Sci. USA, 83:8560-8564 (1986)), the oleosin promoter (Hong et al.,Plant Mol. Biol., 34(3):549-555 (1997)), and zein promoters, such as the15 kD zein promoter, the 16 kD zein promoter, 19 kD zein promoter, 22 kDzein promoter and 27 kD zein promoter. Also suitable are the Osgt-1promoter from the rice glutelin-1 gene (Zheng et al., Mol. Cell Biol.,13:5829-5842 (1993)), the beta-amylase promoter, and the barley hordeinpromoter. Other maturing endosperm promoters include the YP0092, PT0676,and PT0708 promoters.

iv. Ovary Tissue Promoters

Promoters that are active in ovary tissues such as the ovule wall andmesocarp can also be useful, e.g., a polygalacturonidase promoter, thebanana TRX promoter, the melon actin promoter, YP0396, and PT0623.Examples of promoters that are active primarily in ovules includeYP0007, YP0111, YP0092, YP0103, YP0028, YP0121, YP0008, YP0039, YP0115,YP0119, YP0120, and YP0374.

v. Embryo Sac/Early Endosperm Promoters

To achieve expression in embryo sac/early endosperm, regulatory regionscan be used that are active in polar nuclei and/or the central cell, orin precursors to polar nuclei, but not in egg cells or precursors to eggcells. Most suitable are promoters that drive expression only orpredominantly in polar nuclei or precursors thereto and/or the centralcell. A pattern of transcription that extends from polar nuclei intoearly endosperm development can also be found with embryo sac/earlyendosperm-preferential promoters, although transcription typicallydecreases significantly in later endosperm development during and afterthe cellularization phase. Expression in the zygote or developing embryotypically is not present with embryo sac/early endosperm promoters.

Promoters that may be suitable include those derived from the followinggenes: Arabidopsis viviparous-1 (see, GenBank No. U93215); Arabidopsisatmycl (see, Urao, Plant Mol. Biol., 32:571-57 (1996); Conceicao, Plant,5:493-505 (1994)); Arabidopsis FIE (GenBank No. AF129516); ArabidopsisMEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Pat. No.6,906,244). Other promoters that may be suitable include those derivedfrom the following genes: maize MAC1 (see, Sheridan, Genetics,142:1009-1020 (1996)); maize Cat3 (see, GenBank No. L05934; Abler, PlantMol. Biol., 22:10131-1038 (1993)). Other promoters include the followingArabidopsis promoters: YP0039, YP0101, YP0102, YP0110, YP0117, YP0119,YP0137, DME, YP0285, and YP0212. Other promoters that may be usefulinclude the following rice promoters: p530c10, pOsFIE2-2, pOsMEA,pOsYp102, and pOsYp285.

vi. Embryo Promoters

Regulatory regions that preferentially drive transcription in zygoticcells following fertilization can provide embryo-preferentialexpression. Most suitable are promoters that preferentially drivetranscription in early stage embryos prior to the heart stage, butexpression in late stage and maturing embryos is also suitable.Embryo-preferential promoters include the barley lipid transfer protein(Ltp1) promoter (Plant Cell Rep 20:647-654 (2001)), YP0097, YP0107,YP0088, YP0143, YP0156, PT0650, PT0695, PT0723, PT0838, PT0879, andPT0740.

vii. Photosynthetic Tissue Promoters

Promoters active in photosynthetic tissue confer transcription in greentissues such as leaves and stems. Most suitable are promoters that driveexpression only or predominantly in such tissues. Examples of suchpromoters include the ribulose-1,5-bisphosphate carboxylase (RbcS)promoters such as the RbcS promoter from eastern larch (Larix laricina),the pine cab6 promoter (Yamamoto et al., Plant Cell Physiol., 35:773-778(1994)), the Cab-1 promoter from wheat (Fejes et al., Plant Mol. Biol.,15:921-932 (1990)), the CAB-1 promoter from spinach (Lubberstedt et al.,Plant Physiol., 104:997-1006 (1994)), the cab1R promoter from rice (Luanet al., Plant Cell, 4:971-981 (1992)), the pyruvate orthophosphatedikinase (PPDK) promoter from corn (Matsuoka et al., Proc. Natl. Acad.Sci. USA, 90:9586-9590 (1993)), the tobacco Lhcb1*2 promoter (Cerdan etal., Plant Mol. Biol., 33:245-255 (1997)), the Arabidopsis thaliana SUC2sucrose-H+ symporter promoter (Truernit et al., Planta, 196:564-570(1995)), and thylakoid membrane protein promoters from spinach (psaD,psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS). Other photosynthetic tissuepromoters include PT0535, PT0668, PT0886, YP0144, YP0380 and PT0585.

viii. Vascular Tissue Promoters

Examples of promoters that have high or preferential activity invascular bundles include YP0087, YP0093, YP0108, YP0022, and YP0080.Other vascular tissue-preferential promoters include the glycine-richcell wall protein GRP 1.8 promoter (Keller and Baumgartner, Plant Cell,3(10):1051-1061 (1991)), the Commelina yellow mottle virus (CoYMV)promoter (Medberry et al., Plant Cell, 4(2):185-192 (1992)), and therice tungro bacilliform virus (RTBV) promoter (Dai et al., Proc. Natl.Acad. Sci. USA, 101(2):687-692 (2004)).

ix. Inducible Promoters

Inducible promoters confer transcription in response to external stimulisuch as chemical agents or environmental stimuli. For example, induciblepromoters can confer transcription in response to hormones such asgiberellic acid or ethylene, or in response to light or drought.Examples of drought-inducible promoters include YP0380, PT0848, YP0381,YP0337, PT0633, YP0374, PT0710, YP0356, YP0385, YP0396, YP0388, YP0384,PT0688, YP0286, YP0377, PD1367, and PD0901. Examples ofnitrogen-inducible promoters include PT0863, PT0829, PT0665, and PT0886.Examples of shade-inducible promoters include PR0924 and PT0678. Anexample of a promoter induced by salt is rd29A (Kasuga et al. (1999)Nature Biotech 17: 287-291).

x. Basal Promoters

A basal promoter is the minimal sequence necessary for assembly of atranscription complex required for transcription initiation. Basalpromoters frequently include a “TATA box” element that may be locatedbetween about 15 and about 35 nucleotides upstream from the site oftranscription initiation. Basal promoters also may include a “CCAAT box”element (typically the sequence CCAAT) and/or a GGGCG sequence, whichcan be located between about 40 and about 200 nucleotides, typicallyabout 60 to about 120 nucleotides, upstream from the transcription startsite.

xi. Stem Promoters

A stem promoter may be specific to one or more stem tissues or specificto stem and other plant parts. Stem promoters may have high orpreferential activity in, for example, epidermis and cortex, vascularcambium, procambium, or xylem. Examples of stem promoters include YP0018which is disclosed in US20060015970 and CryIA(b) and CryIA(c) (Braga etal. 2003, Journal of New Seeds 5:209-221).

xii. Other Promoters

Other classes of promoters include, but are not limited to,shoot-preferential, callus-preferential, trichome cell-preferential,guard cell-preferential such as PT0678, tuber-preferential, parenchymacell-preferential, and senescence-preferential promoters. In someembodiments, a promoter may preferentially drive expression inreproductive tissues (e.g., PO2916 promoter, SEQ ID NO:31 in61/364,903). Promoters designated YP0086, YP0188, YP0263, PT0758,PT0743, PT0829, YP0119, and YP0096, as described in the above-referencedpatent applications, may also be useful.

xiii. Other Regulatory Regions

A 5′ untranslated region (UTR) can be included in nucleic acidconstructs described herein. A 5′ UTR is transcribed, but is nottranslated, and lies between the start site of the transcript and thetranslation initiation codon and may include the +1 nucleotide. A 3′ UTRcan be positioned between the translation termination codon and the endof the transcript. UTRs can have particular functions such as increasingmRNA stability or attenuating translation. Examples of 3′ UTRs include,but are not limited to, polyadenylation signals and transcriptiontermination sequences, e.g., a nopaline synthase termination sequence.

It will be understood that more than one regulatory region may bepresent in a recombinant polynucleotide, e.g., introns, enhancers,upstream activation regions, transcription terminators, and inducibleelements. Thus, for example, more than one regulatory region can beoperably linked to the sequence of a polynucleotide encoding a biomasscomposition-modulating polypeptide.

Regulatory regions, such as promoters for endogenous genes, can beobtained by chemical synthesis or by subcloning from a genomic DNA thatincludes such a regulatory region. A nucleic acid comprising such aregulatory region can also include flanking sequences that containrestriction enzyme sites that facilitate subsequent manipulation.

IV. TRANSGENIC PLANTS AND PLANT CELLS

A. Transformation

The invention also features transgenic plant cells and plants comprisingat least one recombinant nucleic acid construct described herein. Aplant or plant cell can be transformed by having a construct integratedinto its genome, i.e., can be stably transformed. Stably transformedcells typically retain the introduced nucleic acid with each celldivision. A plant or plant cell can also be transiently transformed suchthat the construct is not integrated into its genome. Transientlytransformed cells typically lose all or some portion of the introducednucleic acid construct with each cell division such that the introducednucleic acid cannot be detected in daughter cells after a sufficientnumber of cell divisions. Both transiently transformed and stablytransformed transgenic plants and plant cells can be useful in themethods described herein.

Transgenic plant cells used in methods described herein can constitutepart or all of a whole plant. Such plants can be grown in a mannersuitable for the species under consideration, either in a growthchamber, a greenhouse, or in a field. Transgenic plants can be bred asdesired for a particular purpose, e.g., to introduce a recombinantnucleic acid into other lines, to transfer a recombinant nucleic acid toother species, or for further selection of other desirable traits.Alternatively, transgenic plants can be propagated vegetatively forthose species amenable to such techniques. As used herein, a transgenicplant also refers to progeny of an initial transgenic plant provided theprogeny inherits the transgene. Seeds produced by a transgenic plant canbe grown and then selfed (or outcrossed and selfed) to obtain seedshomozygous for the nucleic acid construct.

Transgenic plants can be grown in suspension culture, or tissue or organculture. For the purposes of this invention, solid and/or liquid tissueculture techniques can be used. When using solid medium, transgenicplant cells can be placed directly onto the medium or can be placed ontoa filter that is then placed in contact with the medium. When usingliquid medium, transgenic plant cells can be placed onto a flotationdevice, e.g., a porous membrane that contacts the liquid medium. A solidmedium can be, for example, Murashige and Skoog (MS) medium containingagar and a suitable concentration of an auxin, e.g.,2,4-dichlorophenoxyacetic acid (2,4-D), and a suitable concentration ofa cytokinin, e.g., kinetin.

When transiently transformed plant cells are used, a reporter sequenceencoding a reporter polypeptide having a reporter activity can beincluded in the transformation procedure and an assay for reporteractivity or expression can be performed at a suitable time aftertransformation. A suitable time for conducting the assay typically isabout 1-21 days after transformation, e.g., about 1-14 days, about 1-7days, or about 1-3 days. The use of transient assays is particularlyconvenient for rapid analysis in different species, or to confirmexpression of a heterologous biomass composition-modulating polypeptidewhose expression has not previously been confirmed in particularrecipient cells.

Techniques for introducing nucleic acids into monocotyledonous anddicotyledonous plants are known in the art, and include, withoutlimitation, Agrobacterium-mediated transformation, viral vector-mediatedtransformation, electroporation and particle gun transformation, e.g.,U.S. Pat. Nos. 5,538,880; 5,204,253; 6,329,571 and 6,013,863. If a cellor cultured tissue is used as the recipient tissue for transformation,plants can be regenerated from transformed cultures if desired, bytechniques known to those skilled in the art.

B. Screening/Selection

A population of transgenic plants can be screened and/or selected forthose members of the population that have a trait or phenotype conferredby expression of the transgene. For example, a population of progeny ofa single transformation event can be screened for those plants having adesired level of expression of a biomass composition-modulatingpolypeptide or nucleic acid. Physical and biochemical methods can beused to identify expression levels. These include Southern analysis orPCR amplification for detection of a polynucleotide; Northern blots, S1RNase protection, primer-extension, or RT-PCR amplification fordetecting RNA transcripts; enzymatic assays for detecting enzyme orribozyme activity of polypeptides and polynucleotides; and protein gelelectrophoresis, Western blots, immunoprecipitation, and enzyme-linkedimmunoassays to detect polypeptides. Other techniques such as in situhybridization, enzyme staining, and immunostaining also can be used todetect the presence or expression of polypeptides and/orpolynucleotides. Methods for performing all of the referenced techniquesare known. As an alternative, a population of plants comprisingindependent transformation events can be screened for those plantshaving a desired trait, such as a modulated level of biomass. Selectionand/or screening can be carried out over one or more generations, and/orin more than one geographic location. In some cases, transgenic plantscan be grown and selected under conditions which induce a desiredphenotype or are otherwise necessary to produce a desired phenotype in atransgenic plant. In addition, selection and/or screening can be appliedduring a particular developmental stage in which the phenotype isexpected to be exhibited by the plant. Selection and/or screening can becarried out to choose those transgenic plants having a statisticallysignificant difference in a biomass level relative to a control plantthat lacks the transgene. Selected or screened transgenic plants have analtered phenotype as compared to a corresponding control plant, asdescribed in the “Transgenic Plant Phenotypes” section herein.

C. Plant Species

The polynucleotides and vectors described herein can be used totransform a number of monocotyledonous and dicotyledonous plants andplant cell systems, including species from one of the followingfamilies: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae,Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae,Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae,Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae,Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae,Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae,Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae, Plantaginaceae, Poaceae,Rosaceae, Rubiaceae, Salicaceae, Sapindaceae, Solanaceae, Taxaceae,Theaceae, or Vitaceae.

Suitable species may include members of the genus Abelmoschus, Abies,Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon,Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula,Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus,Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum,Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis,Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus,Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea,Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus,Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa,Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia,Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus,Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum,Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale,Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.

Suitable species include Panicum spp., Sorghum spp., Miscanthus spp.,Saccharum spp., Erianthus spp., Populus spp., Andropogon gerardii (bigbluestem), Pennisetum purpureum (elephant grass), Phalaris arundinacea(reed canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea(tall fescue), Spartina pectinata (prairie cord-grass), Medicago sativa(alfalfa), Arundo donax (giant reed), Secale cereale (rye), Salix spp.(willow), Eucalyptus spp. (eucalyptus), Triticosecale (triticum—wheat Xrye) and bamboo.

Suitable species also include Helianthus annuus (sunflower), Carthamustinctorius (safflower), Jatropha curcas (jatropha), Ricinus communis(castor), Elaeis guineensis (palm), Linum usitatissimum (flax), andBrassica juncea.

Suitable species also include Beta vulgaris (sugarbeet), and Manihotesculenta (cassava)

Suitable species also include Lycopersicon esculentum (tomato), Lactucasativa (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato),Brassica oleracea (broccoli, cauliflower, Brussels sprouts), Camelliasinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa),Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus(pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion),Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima(squash), Cucurbita moschata (squash), Spinacea oleracea (spinach),Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), andSolanum melongena (eggplant).

Suitable species also include Papaver somniferum (opium poppy), Papaverorientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabissativa, Camptotheca acuminate, Catharanthus roseus, Vinca rosea,Cinchona officinalis, Colchicum autumnale, Veratrum californica,Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographispaniculata, Atropa belladonna, Datura stomonium, Berberis spp.,Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca,Galanthus wornorii, Scopolia spp., Lycopodium serratum (Huperziaserrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp.,Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis,Chrysanthemum parthenium, Coleus forskohlii, and Tanacetum parthenium.

Suitable species also include Parthenium argentatum (guayule), Heveaspp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixaorellana, and Alstroemeria spp.

Suitable species also include Rosa spp. (rose), Dianthus caryophyllus(carnation), Petunia spp. (petunia) and Poinsettia pulcherrima(poinsettia).

Suitable species also include Nicotiana tabacum (tobacco), Lupinus albus(lupin), Uniola paniculata (oats), bentgrass (Agrostis spp.), Populustremuloides (aspen), Pinus spp. (pine), Abies spp. (fir), Acer spp.(maple), Hordeum vulgare (barley), Poa pratensis (bluegrass), Loliumspp. (ryegrass) and Phleum pratense (timothy).

In some embodiments, a suitable species can be a wild, weedy, orcultivated Pennisetum species such as, but not limited to, Pennisetumalopecuroides, Pennisetum arnhemicum, Pennisetum caffrum, Pennisetumclandestinum, Pennisetum divisum, Pennisetum glaucum, Pennisetumlatifolium, Pennisetum macrostachyum, Pennisetum macrourum, Pennisetumorientale, Pennisetum pedicellatum, Pennisetum polystachion, Pennisetumpolystachion ssp. Setosum, Pennisetum purpureum, Pennisetum setaceum,Pennisetum subangustum, Pennisetum typhoides, Pennisetum villosum, orhybrids thereof (e.g., Pennisetum purpureum x Pennisetum typhoidum).

In some embodiments, a suitable species can be a wild, weedy, orcultivated Miscanthus species and/or variety such as, but not limitedto, Miscanthus x giganteus, Miscanthus sinensis, Miscanthus x ogiformis,Miscanthus floridulus, Miscanthus transmorrisonensis, Miscanthusoligostachyus, Miscanthus nepalensis, Miscanthus sacchariflorus,Miscanthus x giganteus ‘Amuri’, Miscanthus x giganteus ‘Nagara’,Miscanthus x giganteus ‘Illinois’, Miscanthus sinensis var. ‘Goliath’,Miscanthus sinensis var. ‘Roland’, Miscanthus sinensis var. ‘Africa’,Miscanthus sinensis var. ‘Fern Osten’, Miscanthus sinensis var.gracillimus, Miscanthus sinensis var. variegates, Miscanthus sinensisvar. purpurascens, Miscanthus sinensis var. ‘Malepartus’, Miscanthussacchariflorus var. ‘Robusta’, Miscanthus sinensis var. ‘Silberfedher’(aka. Silver Feather), Miscanthus transmorrisonensis, Miscanthuscondensatus, Miscanthus yakushimanum, Miscanthus var. ‘Alexander’,Miscanthus var. ‘Adagio’, Miscanthus var. ‘Autumn Light’, Miscanthusvar. ‘Cabaret’, Miscanthus var. ‘Condensatus’, Miscanthus var.‘Cosmopolitan’, Miscanthus var. ‘Dixieland’, Miscanthus var. ‘GildedTower’ (U.S. Pat. No. PP14,743), Miscanthus var. ‘Gold Bar’ (U.S. Pat.No. PP15,193), Miscanthus var. ‘Gracillimus’, Miscanthus var.‘Graziella’, Miscanthus var. ‘Grosse Fontaine’, Miscanthus var. ‘Hinjoaka Little Nicky’™, Miscanthus var. ‘Juli’, Miscanthus var. ‘Kaskade’,Miscanthus var. ‘Kirk Alexander’, Miscanthus var. ‘Kleine Fontaine’,Miscanthus var. ‘Kleine Silberspinne’ (aka. ‘Little Silver Spider’),Miscanthus var. ‘Little Kitten’, Miscanthus var. ‘Little Zebra’ (U.S.Pat. No. PP13,008), Miscanthus var. ‘Lottum’, Miscanthus var.‘Malepartus’, Miscanthus var. ‘Morning Light’, Miscanthus var.‘Mysterious Maiden’ (U.S. Pat. No. PP16,176), Miscanthus var. ‘Nippon’,Miscanthus var. ‘November Sunset’, Miscanthus var. ‘Parachute’,Miscanthus var. ‘Positano’, Miscanthus var. ‘Puenktchen’(aka ‘LittleDot’), Miscanthus var. ‘Rigoletto’, Miscanthus var. ‘Sarabande’,Miscanthus var. ‘Silberpfeil’ (aka. Silver Arrow), Miscanthus var.‘Silverstripe’, Miscanthus var. ‘Super Stripe’ (U.S. Pat. No. PP18,161),Miscanthus var. ‘Strictus’, or Miscanthus var. ‘Zebrinus’.

In some embodiments, a suitable species can be a wild, weedy, orcultivated sorghum species and/or variety such as, but not limited to,Sorghum almum, Sorghum amplum, Sorghum angustum, Sorghum arundinaceum,Sorghum bicolor (such as bicolor, guinea, caudatum, kafir, and durra),Sorghum brachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghumcontroversum, Sorghum drummondii, Sorghum ecarinatum, Sorghum exstans,Sorghum grande, Sorghum halepense, Sorghum interjectum, Sorghum intrans,Sorghum laxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghummatarankense, Sorghum miliaceum, Sorghum nigrum, Sorghum nitidum,Sorghum plumosum, Sorghum propinquum, Sorghum purpureosericeum, Sorghumstipoideum, Sorghum sudanensese, Sorghum timorense, Sorghumtrichocladum, Sorghum versicolor, Sorghum virgatum, Sorghum vulgare, orhybrids such as Sorghum×almum, Sorghum x sudangrass orSorghum×drummondii.

Thus, the methods and compositions can be used over a broad range ofplant species, including species from the dicot genera Brassica,Carthamus, Glycine, Gossypium, Helianthus, Jatropha, Parthenium,Populus, and Ricinus; and the monocot genera Elaeis, Festuca, Hordeum,Lolium, Oryza, Panicum, Pennisetum, Phleum, Poa, Saccharum, Secale,Sorghum, Triticosecale, Triticum, and Zea. In some embodiments, a plantis a member of the species Panicum virgatum (switchgrass), Sorghumbicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus),Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays(corn), Glycine max (soybean), Brassica napus (canola), Triticumaestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice),Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris(sugarbeet), or Pennisetum glaucum (pearl millet).

In certain embodiments, the polynucleotides and vectors described hereincan be used to transform a number of monocotyledonous and dicotyledonousplants and plant cell systems, wherein such plants are hybrids ofdifferent species or varieties of a specific species (e.g., Saccharumsp. X Miscanthus sp., Sorghum sp. X Miscanthus sp., e.g., Panicumvirgatum x Panicum amarum, Panicum virgatum x Panicum amarulum, andPennisetum purpureum x Pennisetum typhoidum).

D. Transgenic Plant Phenotypes

In some embodiments, a plant in which expression of a biomasscomposition-modulating polypeptide is modulated has increased ordecreased levels of sucrose, ash, or cell wall content. A plant in whichexpression of a biomass composition-modulating polypeptide is modulatedalso can have increased or decreased conversion efficiency. A componentof biomass composition can be increased by at least 2 percent, e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, 55, 60, or more than 60 percent, as compared to thelevel of the biomass component in a corresponding control plant thatdoes not express the transgene. In some embodiments, a plant in whichexpression of a biomass composition-modulating polypeptide is modulatedcan have decreased levels of a biomass component. The level can bedecreased by at least 2 percent, e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30,35, or more than 35 percent, as compared to the level in a correspondingcontrol plant that does not express the transgene.

Increases in a component of biomass composition (e.g., sucrose) in suchplants can provide improved nutritional availability in geographiclocales where intake of plant foods is often insufficient, or for energyproduction (e.g., conversion efficiency). In some embodiments, decreasesin a component of biomass composition in such plants can be useful inenergy production.

In some embodiments, a plant in which expression of a biomasscomposition-modulating polypeptide is modulated can have increased ordecreased levels of a biomass component (e.g., sucrose content) in oneor more plant tissues, e.g., vegetative tissues, reproductive tissues,or root tissues. For example, the level of a biomass component can beincreased by at least 2 percent, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, ormore than 60 percent, as compared to the level in a correspondingcontrol plant that does not express the transgene. In some embodiments,a plant in which expression of a biomass composition-modulatingpolypeptide is modulated can have decreased levels of a biomasscomponent in one or more plant tissues. The level can be decreased by atleast 2 percent, e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or more than35 percent, as compared to the level in a corresponding control plantthat does not express the transgene.

Typically, a difference in the amount of a biomass component in atransgenic plant or cell relative to a control plant or cell isconsidered statistically significant at p≤0.05 with an appropriateparametric or non-parametric statistic, e.g., Chi-square test, Student'st-test, Mann-Whitney test, or F-test. In some embodiments, a differencein the amount of a biomass component is statistically significant atp<0.01, p<0.005, or p<0.001. A statistically significant difference in,for example, the amount of a biomass component in a transgenic plantcompared to the amount of a control plant indicates that the recombinantnucleic acid present in the transgenic plant results in altered biomasscomposition.

The phenotype of a transgenic plant is evaluated relative to a controlplant. A plant is said “not to express” a polypeptide when the plantexhibits less than 10%, e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.5%, 0.1%, 0.01%, or 0.001%, of the amount of polypeptide or mRNAencoding the polypeptide exhibited by the plant of interest. Expressioncan be evaluated using methods including, for example, RT-PCR, Northernblots, S1 RNase protection, primer extensions, Western blots, proteingel electrophoresis, immunoprecipitation, enzyme-linked immunoassays,chip assays, and mass spectrometry. It should be noted that if apolypeptide is expressed under the control of a tissue-preferential orbroadly expressing promoter, expression can be evaluated in the entireplant or in a selected tissue. Similarly, if a polypeptide is expressedat a particular time, e.g., at a particular time in development or uponinduction, expression can be evaluated selectively at a desired timeperiod.

Biomass can include harvestable plant tissues such as leaves, stems, andreproductive structures, or all plant tissues such as leaves, stems,roots, and reproductive structures. In some embodiments, biomassencompasses only above ground plant parts. In some embodiments, biomassencompasses only stem plant parts. In some embodiments, biomassencompasses only above ground plant parts except inflorescence and seedparts of a plant. Biomass can be measured as described in the examplessection. Biomass can be quantified as dry matter yield, which is themass of biomass produced (usually reported in T/acre) if thecontribution of water is subtracted from the fresh mater weight. Drymatter yield (DMY) yield is calculated using the fresh matter weight(FMW) and a measurement of weight percent moisture (M) in the followingequation. DMY=((100−M)/100)*FMW. Biomass can be quantified as freshmatter yield, which is the mass of biomass produced (usually reported inT/acre) on an as-received basis, which includes the weight of moisture.

V. MODIFYING ENDOGENOUS NUCLEIC ACIDS ENCODING BIOMASSCOMPOSITION-MODULATING POLYPEPTIDES

This document also features plant cells and plants in which anendogenous biomass composition-modulating nucleic acid described hereinhas been modified (e.g., a regulatory region, intron, or coding regionof the biomass composition-modulating nucleic acid has been modified).The biomass composition of such plants is altered relative to thecorresponding composition of a control plant in which the endogenousnucleic acid is not modified. Such plants are referred to herein asmodified plants and may be used to produce, for example, increasedamounts of a biomass component (e.g., sucrose).

Endogenous nucleic acid can be modified by homologous recombinationtechniques. For example, sequence specific endonucleases (e.g., zincfinger nucleases (ZFNs)) and meganucleases can be used to stimulatehomologous recombination at endogenous plant genes. See, e.g., Townsendet al., Nature 459:442-445 (2009); Tovkach et al., Plant J., 57:747-757(2009); and Lloyd et al., Proc. Natl. Acad. Sci. USA, 102:2232-2237(2005). In particular, ZFNs engineered to create DNA double strandbreaks at specific loci can be used to make targeted sequence changes inendogenous plant genes. For example, an endogenous plant gene can bereplaced with a variant containing one or more mutations (e.g., producedusing site-directed mutagenesis or directed evolution). In someembodiments, site directed mutagenesis is achieved via non-homologousend joining such that after breaking DNA, endogenous DNA repairmechanisms ligate the break, often introducing slight deletions oradditions that can be screened at the cell or plant level for desiredphenotypes. Moore and Haber, Mol Cell Biol., 16(5):2164-73 (1996).

In some embodiments, endogenous nucleic acids can be modified bymethylation or demethylation such that the expression of the modifiedendogenous nucleic acid is altered. For example, a double stranded RNAcan be used to activate gene expression by targeting noncodingregulatory regions in gene promoters. See Shibuya et al., Proc Natl AcadSci USA, 106(5): 1660-1665 (2009); and Li et al., Proc Natl Acad SciUSA, 103(46):17337-42 (2006). In some embodiments, ZFNs engineered tocreate DNA double strand breaks at specific loci can be used to insert aDNA fragment having at least one region that overlaps with theendogenous DNA to facilitate homologous recombination, such that thenon-overlapping portion of the DNA fragment is integrated at the breaksite. For example, a fragment can be inserted into an endogenouspromoter and/or regulatory region at a specific site where a ZFN createda double stranded break to alter the expression of an endogenous gene.For example, a fragment that is inserted into an endogenous gene codingregion at a specific site where a ZFN created a double stranded breakcan result in expression of a chimeric gene. For example, a fragmentthat functions as a regulatory region or promoter that is inserted intoan endogenous DNA region immediately upstream of a gene coding sequenceat a specific site where a ZFN creates a double strand break can resultin altered expression of the endogenous gene.

In some embodiments, endogenous nucleic acids can be modified usingactivation tagging. For example, a vector containing multiple copies ofan enhancer element from the constitutively active promoter of thecauliflower mosaic virus (CaMV) 35S gene can be used to activate anendogenous gene. See, Weigel et al., Plant Physiology, 122:1003-1013(2000).

In some embodiments, endogenous nucleic acids can be modified byintroducing an engineered transcription activation/repression factor(e.g., zinc finger protein transcription factor, or ZFP TF. See, forexample, the world wide web atsangamo.com/tech/tech_plat_over.html#whatarezfp). For example, asynthetic transcription facto sequence of a zinc finger DNA bindingdomain and a VP16 activation domain can be designed to bind to aspecific endogenous DNA site and alter expression of an endogenous gene.An engineered transcription activation/repression factor (such as ZFPTF) can activate, repress, or switch the target endogenous biomass,sucrose, and/or conversion-gene expression by binding specifically tothe promoter region or coding region of the endogenous gene. Engineerednucleases that cleave specific DNA sequences in vivo can also bevaluable reagents for targeted mutagenesis. One such class ofsequence-specific nucleases can be created by fusing transcriptionactivator-like effectors (TALEs) to the catalytic domain of the Foldendonuclease. Both native and custom TALE-nuclease fusions direct DNAdouble-strand breaks to specific, targeted sites. Christian, et al.,Genetics 186: 757-761 (2010).

In some embodiments, endogenous nucleic acids can be modified bymutagenesis. Genetic mutations can be introduced within regenerableplant tissue using one or more mutagenic agents. Suitable mutagenicagents include, for example, ethyl methane sulfonate (EMS),N-nitroso-N-ethylurea (ENU), methyl N-nitrosoguanidine (MNNG), ethidiumbromide, diepoxybutane, ionizing radiation, x-rays, UV rays and othermutagens known in the art. Suitable types of mutations include, forexample, insertions or deletions of nucleotides, and transitions ortransversions in the endogenous nucleic acid sequence. In oneembodiment, TILLING (Targeted Induced Local Lesions In Genomes) can beused to produce plants having a modified endogenous nucleic acid.TILLING combines high-density mutagenesis with high-throughput screeningmethods. See, for example, McCallum et al., Nat Biotechnol 18: 455-457(2000); reviewed by Stemple, Nat Rev Genet 5(2):145-50 (2004).

In some embodiments, an endogenous nucleic acid can be modified via agene silencing technique. See, for example, the section herein regarding“Inhibition of Expression of a Biomass composition-modulatingPolypeptide.”

A population of plants can be screened and/or selected for those membersof the population that have a modified nucleic acid. A population ofplants also can be screened and/or selected for those members of thepopulation that have a trait or phenotype conferred by expression of themodified nucleic acid. As an alternative, a population of plants can bescreened for those plants having a desired trait, such as a modulatedlevel of biomass. For example, a population of progeny can be screenedfor those plants having a desired level of expression of a biomasscomposition-modulating polypeptide or nucleic acid. Physical andbiochemical methods can be used to identify modified nucleic acidsand/or expression levels as described with transgenic plants. Selectionand/or screening can be carried out over one or more generations, and/orin more than one geographic location. In some cases, plants can be grownand selected under conditions which induce a desired phenotype or areotherwise necessary to produce a desired phenotype in a modified plant.In addition, selection and/or screening can be applied during aparticular developmental stage in which the phenotype is expected to beexhibited by the plant. Selection and/or screening can be carried out tochoose those modified plants having a statistically significantdifference in biomass composition relative to a control plant in whichthe nucleic acid has not been modified. Selected or screened modifiedplants have an altered phenotype as compared to a corresponding controlplant, as described in the “Transgenic Plant Phenotypes” section herein.

Although a plant or plant cell in which an endogenous biomasscomposition-modulating nucleic acid has been modified is not transgenicfor that particular nucleic acid, it will be appreciated that such aplant or cell may contain transgenes. For example, a modified plant cancontain a transgene for other traits, such as herbicide tolerance orinsect resistance. As another example, a modified plant can contain oneor more transgenes that, in conjunction with modifications of one ormore endogenous nucleic acids, exhibits an increase in a component ofbiomass.

As with transgenic plant cells, modified plant cells can constitute partor all of a whole plant. Such plants can be grown in the same manner asdescribed for transgenic plants and can be bred or propagated in thesame manner as described for transgenic plants.

VI. PLANT BREEDING

Genetic polymorphisms that are useful in such methods include simplesequence repeats (SSRs, or microsatellites), rapid amplification ofpolymorphic DNA (RAPDs), single nucleotide polymorphisms (SNPs),amplified fragment length polymorphisms (AFLPs) and restriction fragmentlength polymorphisms (RFLPs). SSR polymorphisms can be identified, forexample, by making sequence specific probes and amplifying template DNAfrom individuals in the population of interest by PCR. For example, PCRtechniques can be used to enzymatically amplify a genetic markerassociated with a nucleotide sequence conferring a specific trait (e.g.,nucleotide sequences described herein). PCR can be used to amplifyspecific sequences from DNA as well as RNA, including sequences fromtotal genomic DNA or total cellular RNA. When using RNA as a source oftemplate, reverse transcriptase can be used to synthesize complementaryDNA (cDNA) strands. Various PCR methods are described, for example, inPCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., ColdSpring Harbor Laboratory Press, 1995.

Generally, sequence information from polynucleotides flanking the regionof interest or beyond is employed to design oligonucleotide primers thatare identical or similar in sequence to opposite strands of the templateto be amplified. Primers are typically 14 to 40 nucleotides in length,but can range from 10 nucleotides to hundreds of nucleotides in length.Template and amplified DNA is repeatedly denatured at a high temperatureto separate the double strand, then cooled to allow annealing of primersand the extension of nucleotide sequences through the microsatellite,resulting in sufficient DNA for detection of PCR products. If the probesflank an SSR in the population, PCR products of different sizes will beproduced. See, e.g., U.S. Pat. No. 5,766,847.

PCR products can be qualitative or quantitatively analyzed using severaltechniques. For example, PCR products can be stained with a fluorescentmolecule (e.g., PicoGreen® or OliGreen®) and detected in solution usingspectrophotometry or capillary electrophoresis. In some cases, PCRproducts can be separated in a gel matrix (e.g., agarose orpolyacrylamide) by electrophoresis, and size-fractionated bandscomprising PCR products can be visualized using nucleic acid stains.Suitable stains can fluoresce under UV light (e.g., Ethidium bromide, GRSafe, SYBR® Green, or SYBR® Gold). The results can be visualized viatransillumination or epi-illumination, and an image of the fluorescentpattern can be acquired using a camera or scanner, for example. Theimage can be processed and analyzed using specialized software (e.g.,ImageJ) to measure and compare the intensity of a band of interestagainst a standard loaded on the same gel.

Alternatively, SSR polymorphisms can be identified by using PCRproduct(s) as a probe against Southern blots from different individualsin the population. See, Refseth et al., (1997) Electrophoresis 18: 1519.Briefly, PCR products are separated by length through gelelectrophoresis and transferred to a membrane. SSR-specific DNA probes,such as oligonucleotides labeled with radioactive, fluorescent, orchromogenic molecules, are applied to the membrane and hybridize tobound PCR products with a complementary nucleotide sequence. The patternof hybridization can be visualized by autoradiography or by developmentof color on the membrane, for example.

In some cases, PCR products can be quantified using a real-timethermocycler detection system. For example, Quantitative real-time PCRcan use a fluorescent dye that forms a DNA-dye-complex (e.g., SYBR®Green), or a fluorophore-containing DNA probe, such as single-strandedoligonucleotides covalently bound to a fluorescent reporter orfluorophore (e.g. 6-carboxyfluorescein or tetrachlorofluorescin) andquencher (e.g., tetramethylrhodamine or dihydrocyclopyrroloindoletripeptide minor groove binder). The fluorescent signal allows detectionof the amplified product in real time, thereby indicating the presenceof a sequence of interest, and allowing quantification of the copynumber of a sequence of interest in cellular DNA or expression level ofa sequence of interest from cellular mRNA.

The identification of RFLPs is discussed, for example, in Alonso-Blancoet al. (Methods in Molecular Biology, vol. 82, “Arabidopsis Protocols”,pp. 137-146, J. M. Martinez-Zapater and J. Salinas, eds., c. 1998 byHumana Press, Totowa, N.J.); Burr (“Mapping Genes with RecombinantInbreds”, pp. 249-254, in Freeling, M. and V. Walbot (Ed.), The MaizeHandbook, c. 1994 by Springer-Verlag New York, Inc.: New York, N.Y.,USA; Berlin Germany; Burr et al. Genetics (1998) 118: 519; and Gardiner,J. et al., (1993) Genetics 134: 917). For example, to produce a RFLPlibrary enriched with single- or low-copy expressed sequences, total DNAcan be digested with a methylation-sensitive enzyme (e.g., PstI). Thedigested DNA can be separated by size on a preparative gel.Polynucleotide fragments (500 to 2000 bp) can be excised, eluted andcloned into a plasmid vector (e.g., pUC18). Southern blots of plasmiddigests can be probed with total sheared DNA to select clones thathybridize to single- and low-copy sequences. Additional restrictionendonucleases can be tested to increase the number of polymorphismsdetected.

The identification of AFLPs is discussed, for example, in EP 0 534 858and U.S. Pat. No. 5,878,215. In general, total cellular DNA is digestedwith one or more restriction enzymes. Restriction halfsite-specificadapters are ligated to all restriction fragments and the fragments areselectively amplified with two PCR primers that have correspondingadaptor and restriction site specific sequences. The PCR products can bevisualized after size-fractionation, as described above.

In some embodiments, the methods are directed to breeding a plant line.Such methods use genetic polymorphisms identified as described above ina marker assisted breeding program to facilitate the development oflines that have a desired alteration in biomass composition. Once asuitable genetic polymorphism is identified as being associated withvariation for the trait, one or more individual plants are identifiedthat possess the polymorphic allele correlated with the desiredvariation. Those plants are then used in a breeding program to combinethe polymorphic allele with a plurality of other alleles at other locithat are correlated with the desired variation. Techniques suitable foruse in a plant breeding program are known in the art and include,without limitation, backcrossing, mass selection, pedigree breeding,bulk selection, crossing to another population and recurrent selection.These techniques can be used alone or in combination with one or moreother techniques in a breeding program. Thus, each identified plants isselfed or crossed a different plant to produce seed which is thengerminated to form progeny plants. At least one such progeny plant isthen selfed or crossed with a different plant to form a subsequentprogeny generation. The breeding program can repeat the steps of selfingor outcrossing for an additional 0 to 5 generations as appropriate inorder to achieve the desired uniformity and stability in the resultingplant line, which retains the polymorphic allele. In most breedingprograms, analysis for the particular polymorphic allele will be carriedout in each generation, although analysis can be carried out inalternate generations if desired.

In some cases, selection for other useful traits is also carried out,e.g., selection for fungal resistance or bacterial resistance. Selectionfor such other traits can be carried out before, during or afteridentification of individual plants that possess the desired polymorphicallele.

VII. ARTICLES OF MANUFACTURE

Transgenic plants provided herein have various uses in the agriculturaland energy production industries. For example, transgenic plantsdescribed herein can be used to make animal feed and food products. Suchplants, however, are often particularly useful as a feedstock for energyproduction.

Transgenic plants described herein often produce higher yields of grainand/or biomass per hectare, relative to control plants that lack theexogenous nucleic acid. In some embodiments, such transgenic plantsprovide equivalent or even increased yields of grain and/or biomass perhectare relative to control plants when grown under conditions ofreduced inputs such as fertilizer and/or water. Thus, such transgenicplants can be used to provide yield stability at a lower input costand/or under environmentally stressful conditions such as drought. Insome embodiments, plants described herein have a composition thatpermits more efficient processing into free sugars, and subsequentlyethanol, for energy production. In some embodiments, such plants providehigher yields of ethanol, butanol, dimethyl ether, other biofuelmolecules, and/or sugar-derived co-products per kilogram of plantmaterial, relative to control plants. Such processing efficiencies arebelieved to be derived from the composition of the plant material,including, but not limited to, content of glucan, cellulose,hemicellulose, and lignin. By providing higher biomass yields at anequivalent or even decreased cost of production, the transgenic plantsdescribed herein improve profitability for farmers and processors aswell as decrease costs to consumers.

Seeds from transgenic plants described herein can be conditioned andbagged in packaging material by means known in the art to form anarticle of manufacture. Packaging material such as paper and cloth arewell known in the art. A package of seed can have a label, e.g., a tagor label secured to the packaging material, a label printed on thepackaging material, or a label inserted within the package, thatdescribes the nature of the seeds therein.

VIII. USES AND ADVANTAGES

Sorghum plants described herein can be grown in large fields (e.g., 50to 10,000 acre fields) to obtain harvestable biomass. For example, thesorghum plants provided herein can be grown in fields of 100 acres ormore at locations suitable for sorghum growth such as southern UnitedStates, Brazil, and Mexico.

In one embodiment, the stalks of sorghum plants described herein areharvested and processed, e.g., extracted using pressing and/or millingtechniques, to obtain sorghum stem juice. For example, the stalks can beharvested by hand or mechanical harvesters, and then crushed and pressedwith a horizontal or vertical mill to extract the juice. One objectiveof the pressing and/or milling processes is to extract the largestpossible amount of juice from the sorghum biomass. Another objective isto produce bagasse with a low moisture content to be burned as a boilerfuel for electricity generation, thereby allowing a production plant tobe self-sufficient in energy.

Sucrose, i.e., table sugar, can be produced from the juice usingtechniques including filtering, clarifying, decolorizing, and repeatedconcentration and crystallization. In some embodiments, table sugar isproduced by blending sweet sorghum juice with sugarcane juice prior tocrystallization, thereby increasing the total yield of table sugar.

In other embodiments, the sugars in the juice can be fermented toproduce a biofuel. For example, the juice can be filtered and used in afermentation reaction to produce a biofuel. Examples of biofuelsinclude, without limitation, biodiesel, methanol, ethanol, butanol,linear alkanes (C5-C20), branched-chain alkanes (C5-C26), mixed alkanes,linear alcohols (C1-C20), branched-chain alcohols (C1-C26), linearcarboxylic acids (C2-C20), and branched-chain carboxylic acids (C2-C26).In some cases, the methods and materials provided herein can be used tomake other chemical compounds such as ethers, esters, and amides of theaforementioned acids and alcohols, as well as other conjugates of thesechemicals. In some cases, one or more of these compounds can bechemically converted into other high value and/or high volume chemicals.

Any appropriate microorganism can be used to produce biofuel in afermentation reaction. For example, one or more microorganisms designedto produce ethanol can be used in fermentation reactions with sorghumjuice to produce ethanol-containing reaction products. In some cases, amicroorganism useful for producing one or more biofuels as describedherein is from a genus such as Clostridium, Zymomonas, Escherichia,Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus,Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter,Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, andSaccharomyces. For example, ethanologenic yeast can be used in afermentation reaction containing sorghum juice to produce ethanol.

Any appropriate fermentation process can be used to produce biofuelusing sorghum juice. For example, batch, fed-batch, or continuousfermentation processes can be used to produce a biofuel using sorghumjuice. A batch fermentation process can include adding sorghum juicesubstrate, fermentation organism(s) and culture medium at the beginningof the fermentation and not replenishing once fermentation has begun. Insome cases, one or more culture parameters, e.g., pH and oxygenconcentration, are monitored and adjusted during the fermentationprocess.

In some cases, a fed-batch fermentation process can be used to producebiofuel using sorghum juice obtained from sorghum plants providedherein. A fed-batch fermentation process is similar to a batchfermentation process except that substrate is added, and optionallyculture medium nutrients, at intervals as fermentation progresses. Insome cases, one or more culture parameters, e.g., pH, dissolved oxygenconcentration, and/or carbon dioxide to oxygen ratio, are monitored andadjusted during the fermentation process. Fed-batch fermentationprocesses can allow users to control the amount of substrate within thefermentation reaction.

Continuous fermentation processes also can be used to produce biofuelusing sorghum juice obtained from sorghum plants provided herein. Acontinuous fermentation process can be an open system in which a definedfermentation medium containing sorghum juice material is continuouslyadded to a bioreactor and an amount (e.g., an equal amount) ofconditioned media is continuously removed for subsequent processing.Continuous fermentation can often be performed such that thefermentation organism is maintained at a high cell density and in aprolonged exponential growth phase, resulting in higher productivitythan batch fermentation.

Examples of batch, fed-batch, and continuous fermentation processes thatcan be used to produce biofuel using sorghum juice obtained from plantsprovided herein are described elsewhere (Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, Second Edition(1989) Sinauer Associates, Inc., Sunderland, Mass.; and Deshpande,Mukund V., Appl. Biochem. Biotechnol., 36:227 (1992)).

Any appropriate fermentation media containing sorghum juice can be usedin a fermentation reaction to produce biofuel. In some cases,fermentation media used to produce biofuel as described herein cancontain sorghum juice as the primary carbon source (e.g., primary sourceof glucose, fructose, sucrose, mannose, or other sugars). In some cases,one or more other carbon sources can be used in combination with sorghumjuice provided herein to form fermentation media for producing biofuel.For example, sorghum juice obtained from sorghum plants provided hereincan be combined with sugarcane juice (garapa) to form fermentation mediafor producing biofuel. In some cases, one or more other components suchas minerals, salts, cofactors, and buffers can be included withinfermentation media to promote culture growth and/or biofuel production.Examples of commercially available broths that can be used incombination with sorghum juice material to create fermentation mediainclude, without limitation, Luria Bertani (LB) broth, SabouraudDextrose (SD) broth, and Yeast medium (YM) broth.

Any appropriate culture conditions can be used to perform fermentationreactions designed to produce biofuel using sorghum juice. For example,fermentation cultures can be grown or maintained at a temperature in therange of about 25° C. to about 40° C. and at a pH in the range of pH 5.0to pH 9.0 (e.g., a pH in the range of 6.0 and 8.0, of 6.5 and 7.5, or6.5 and 7.0). A fermentation reaction can be performed under aerobic,microaerobic, or anaerobic conditions.

In some cases, biofuel production can be monitored during a fermentationreaction or can be assessed when the fermentation reaction is completed.Any appropriate method can be used to assess biofuel production. Forexample, high performance liquid chromatography (HPLC) or gaschromatography (GC) can be used to measure biofuel production.

Once produced, biofuel can be isolated from the fermentation product.For example, techniques such as centrifugation, filtration, decantation,or combinations thereof can be performed to remove solids from thefermentation product. Once most or all of the solid material is removed,biofuel present within the remaining material can be isolated by, forexample, techniques such as distillation, liquid-liquid extraction,dehydration, membrane-based separation, or combinations thereof. In somecases, molecular sieves, distillation techniques, azeotropicdistillation techniques, centrifugation, vacuum distillation, orcombinations thereof can be used to separate biofuel (e.g., ethanol)from water and/or fermentation byproducts. For example, water can beremoved from an azeotropic ethanol/water mixture obtained from afermentation reaction by azeotropic distillation to result in hydrousethanol having about 95 to about 96.5 percent ethanol and about 3.5 toabout 5 percent water. Azeotropic distillation can include addingbenzene or cyclohexane to an ethanol/water mixture. When thesecomponents are added to the mixture, they can form a heterogeneousazeotropic mixture in vapor-liquid-liquid equilibrium. This can bedistilled to produce anhydrous ethanol at the bottom of a column and avapor mixture of water and cyclohexane/benzene. When condensed, thematerial can become a two-phase liquid mixture. In some cases, anextractive distillation process that involves adding a ternary componentthat increases the volatility of ethanol can be performed. Distillationof the ternary mixture can result in anhydrous ethanol on the top streamof a column.

In some cases, dehydration methods such as those involving molecularsieve techniques can be used to remove water from a biofuel. Forexample, ethanol vapor under pressure can be passed through a bed ofmolecular sieve beads. The pore size of the beads can be designed toallow absorption of water while excluding ethanol. After a period oftime, the bed can be regenerated under vacuum or through the flow ofinert gas (e.g., N2) to remove absorbed water. In some cases, two ormore beds of beads can be used. In such cases, one can be used to absorbwater, while the other one is undergoing regeneration. In some cases,the use of molecular sieve techniques can be performed in a manner thatdoes not involve the use of distillation techniques.

In some cases, production of ethanol for biofuel involves denaturationof the ethanol. Ethanol can be denatured by, for example, combining itwith natural gasoline, unleaded gasoline, or gasoline blend stocks.Corrosion inhibitors such as Ashland Amergy ECI-6 or Petrolite Tolad3222 can be added to fuel ethanol if desired. Ethanol for fuel use canmeet the specifications of ASTM D4806 (e.g., ASTM D4806-09). In somecases, the ethanol meets the specifications of ASTM D5453-93 for sulfurcontent, the specifications of ASTM D5580-95 for benzene or aromaticcontent, and/or the specifications of ASTM D6550-00 for olefin content.In some cases, ethanol for fuel use, produced as described herein, canmeet Brazilian specification ANP#36 for hydrous ethanol or anhydrousethanol.

In some cases, biomass remaining after extraction of juice (e.g.,bagasse such as low moisture bagasse) or biomass not used for juiceextraction can be used as a source of cellulosic material. Suchcellulosic material can be used in fermentation reactions designed tometabolize cellulose and/or other sorghum biomolecules in order toproduce biofuel or can be used in combustion reactions designed toproduce heat for use in energy production.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

IX. EXAMPLES Example 1 Procedures for Conversion Analysis

The conversion efficiency of control and transgenic switchgrass lineswas determined indirectly using NIR composition and conversion modelsfor switchgrass. See, WO2009/059176. Samples were prepared for analysisby drying the tissue samples for at least 3 days in an incubator set at45° C. Dried tissues were milled using a Wiley Mill fitted with 20-meshfilter. Milled samples contained in a vial were scanned three times. Theaverage scan was run through the NIR model and the predictedpretreatment liquid (PL) and saccharification (SAC) values weredetermined accordingly.

The yield of conversion was directly calculated as follows: [PLNvalue+SAC value]/amount of biomass weight, wherein “PLN” refers topretreatment liquor neutralization, and “SAC” refers to the sugar valuefrom the saccrification analysis. The following procedures were used toobtain the PLN and SAC values.

Microwave pretreatment: Milled tissues were weighed to obtainapproximately 0.025 g. The moisture content of the weighed tissues wasdetermined using the Denver Moisture Content analyzer. Tissues weretransferred into separate Biotage microwave vials that were previouslytared. Appropriate volume of sulfuric acid was then added into thesamples to give a final concentration of 1.3%. Samples were pretreatedin the microwave using the following settings: 165° C., 5 minutes, veryhigh absorbance, 2.0-5.0 vial, 600 rpm stir speed (SWAVE default). Thevials with the microwaved samples were centrifuged at 4000 rpm for 5 minwith a deceleration rate set at ≤5. A minimum of 4 ml of PL from eachvial was transferred into pre-labeled 15 ml Corning conical tubes. ThepH of the PL fraction was measured. The PL was kept frozen until readyto analyze. The residue in each vial was washed several times by adding5 ml water followed by centrifugation step at 4000 rpm for 5 min. The pHof the wash was monitored until it reached between 5 and 6 usingappropriate pH indicator strips. The solid fraction was stored forsaccharification analysis.

Pretreatment Liquor Analysis: To determine PLN (neutralized pretreatmentliquor), calcium carbonate was added to an appropriate aliquot of eachPL fraction until its pH reached between 5 and 6. The neutralizedmixture was centrifuged at 4000 rpm for 2 min; after which 2 ml of theneutralized liquor was transferred to storage tubes.

To determine the sugar content, the neutralized fraction (PLN) wasanalyzed using a YSI Sugar Analyzer and/or by HPLC.

Saccharification Analysis: Water was added to the solid fractionobtained from the microwave pretreatment. Appropriate volume of enzymemixture (containing appropriate weight of proprietary enzymes,tetracycline and cyclohexamide in citrate buffer) was added to themixture followed by incubation at 50° C. in a rotating incubator. At theappropriate time period, an aliquot from the reaction was transferred toa microcentrifuge tube. The reaction was stopped by boiling the mixturefor 5 min. The mixture was centrifuged for 2 min at 14,000 rpm. Thesupernatant was taken for sugar analysis using a YSI Sugar Analyzerand/or by HPLC. This sugar value represents the SAC value.

Example 2 Protocol for Sucrose Analysis

The sucrose content of control and transgenic switchgrass lines wasdetermined indirectly using the NIR composition model for switchgrass.See WO2009/059176. Samples were prepared for analysis by drying thetissue samples for at least 3 days in an incubator set at 45° C. Driedtissues were milled using a Wiley Mill fitted with 20-mesh filter.Milled samples contained in a vial were scanned three times. The averagescan was run through the NIR model and the predicted PL and SAC valueswere determined accordingly.

The sucrose content of selected samples was directly analyzed asfollows. An appropriate amount of milled biomass (3-4 g) was placed intocell vial for extraction using the ASE200 extractor. Extraction wasperformed using water as solvent with the extractor set at followingparameters: 1500 psi pressure, 100° C. temperature, no preheating, 5 minramping, 7 min static step, and purging for 2 min. The volume of thecollected extract was measured. Appropriate dilutions of the extractswere run through HPLC analysis to quantify the amount of sucrose usingreference standards. The % sucrose content was calculated as follows:the amount of sucrose divided by the amount of biomass used in theextraction.

Example 3 Transgenic Switchgrass Lines

The following symbols are used in with respect to transformations: T₀:plant regenerated from transformed tissue culture; T₁: first generationprogeny of self-pollinated T₀ plants; T₂: second generation progeny ofself-pollinated T₁ plants; T₃: third generation progeny ofself-pollinated T₂ plants.

The following nucleic acids were isolated from Panicum virgatum plants:CeresClone: 1807011 (SEQ ID NO:1); Ceres Clone 1955550 (SEQ ID NO:64);CeresClone: 240112 (SEQ ID NO:245); CeresClone: 1900192 (SEQ ID NO:279);CeresClone: 1776501 (SEQ ID NO:347); CeresClone: 1804732 (SEQ IDNO:415); CeresClone: 1955550 (SEQ ID NO:640); and CeresClone: 1789981(SEQ ID NO:773).

Each isolated nucleic acid described above was cloned into T-DNA binaryvectors, which were introduced into switchgrass (A26 or A10 clonallypropagated lines) by Agrobacterium-mediated transformation essentiallyas described in Richards et al., Plant Cell. Rep. 20:48-54 (2001) andSomleva et al., Crop Sci. 42:2080-2087 (2002). At least two independentevents from each transformation were selected for further study; theseevents were referred to as switchgrass screening lines. T0 plants weregrown in a greenhouse. The presence of each construct was confirmed byPCR.

Example 4 NIR Conversion Prediction for Transgenic Line PV00467

T₀ tissues from 22 events of PV00467 containing Ceres Clone 1955550 (SEQID NO:64) were analyzed as described in Example 1. Severalnon-transgenic wild-type plants that were regenerated at the same timeas the transgenic plants were used as controls (also called batchwild-type control). The amount of glucose released after acidpretreatment (mg/g) of PV00467 lines is presented in Table 1. Theaverage of the batch wild-type control plants (i.e., wt batch average)and the overall average of different wild-type controls from differentbatches (i.e., wt running average) are also presented in Table 1. Thepredicted glucose released in the pretreated liquor of some of thePV00467 transgenic events was higher as compared to the wild-typecontrols (either using the wt batch average value or the wt runningaverage value).

TABLE 1 Plant Line PLN Glu Rel PV00467-04 65.3 PV00467-05 58.9PV00467-06 82 PV00467-10 59.4 PV00467-11 62.3 PV00467-12 77.7 PV00467-1365.4 PV00467-14 67.7 PV00467-15 62.9 PV00467-19 49.4 PV00467-20 69.2PV00467-21 67.7 PV00467-22 65.2 PV00467-24 53.7 PV00467-26 49.8PV00467-27 52.5 PV00467-28 53.5 PV00467-29 52.6 PV00467-30 74.7PV00467-31 51.3 PV00467-32 60.1 PV00467-36 58.3 WT (Batch) Ave 55.18 WT(Batch) SD 4.90 WT Running Ave 58.33 WT Running SD 9.08

Example 5 NIR Conversion Prediction for Transgenic Line PV00508

To tissues from 25 events of PV00508 containing Ceres Clone 1776501 (SEQID NO:347) were analyzed as described in Example 1. Severalnon-transgenic wild-type plants that were regenerated at the same timeas the transgenic plants were used as controls (also called as batchwild-type control). The amount of glucose released after acidpretreatment (mg/g) of PV00508 lines is presented in Table 2. Theaverage of the batch wild-type control plants (i.e., wt batch average)and the average of different wild-type controls from different batches(i.e., wt running average) are also presented in Table 2. The predictedglucose released in the pretreated liquor of some of the PV00508transgenic events was higher as compared to the wild-type controls(either using the wt batch average value or the wt running averagevalue).

TABLE 2 Plant Line PLN Glu Rel PV00508-02 103.7 PV00508-03 98.9PV00508-04 114.7 PV00508-05 97.7 PV00508-08 109.7 PV00508-09 103.3PV00508-10 98.5 PV00508-12 93.4 PV00508-13 89 PV00508-15 78.2 PV00508-1880.5 PV00508-19 70.6 PV00508-20 84.7 PV00508-21 76 PV00508-22 90.4PV00508-23 92.1 PV00508-24 91.3 PV00508-26 102.1 PV00508-27 84.1PV00508-29 74.4 PV00508-30 86.2 PV00508-31 97.4 PV00508-33 96.8PV00508-34 97.3 PV00508-35 81.3 WT (Batch) Ave 77.05 WT (Batch) SD 12.77WT Running Ave 58.33 WT Running SD 9.08

Example 6 Sucrose Content of Transgenic Lines UAC-20, UAC-22, and UAC-15

T₀ tissues from 5 events of UAC-20 containing Ceres Clone 1900192 (SEQID NO:279), 7 events of UAC-22 containing Ceres Clone 1807011 (SEQ IDNO:1), and 3 events of UAC-15 containing Ceres Clone 1804732 (SEQ IDNO:415) were analyzed as described in Example 2. Further analysis of theevents of UAC-22 indicated that Ceres Clone 1807011 contains a deletionof a nucleotide at position 667 of SEQ ID NO:1, resulting in theproduction of a truncated protein. UAC-FA4 and UAC-NK4K were used ascontrols. UAC-FA4 is a wild-type plant regenerated from callus that wasnot transformed. UAC-NB4K corresponds to plants that were regeneratedfrom callus transformed with an empty vector (i.e., with no insert). Theaverage total sucrose content is presented in Table 3. All seven of theevents of UAC-22 had an increased total sucrose content while three ofthe UAC-20 events and two of the UAC-15 events had an increased totalsucrose content.

TABLE 3 Avg total % SUC StdDev UAC-20-14 9.62 0.15 UAC-20-15 2.55 0.12UAC-20-21 7.85 0.03 UAC-20-5 11.56 0.10 UAC-20-9 9.98 0.01 UAC-22-1010.67 0.41 UAC-22-11 10.11 1.00 UAC-22-14 7.91 0.30 UAC-22-15 5.29 0.19UAC-22-17 11.73 0.63 UAC-22-21 8.41 0.18 UAC-22-26 9.98 0.26 UAC-15-14.35 0.08 UAC-15-3 9.66 1.00 UAC-15-5 6.94 0.08 UAC-FA4-12 1.13 0.04UAC-FA4-12 3.04 0.22 UAC-NB4K-1 4.37 0.06 UAC-NB4K-9 1.46 0.05

Example 7 NIR Conversion Prediction for Transgenic Lines UAC-15, UAC-19,and UAC-22

T₀ tissues from one event of UAC-15 containing Ceres Clone 1804732 (SEQID NO:415), one event of UAC-19 containing Ceres Clone 1789981 (SEQ IDNO:773), and one event of UAC-22 containing Ceres Clone 1807011 (SEQ IDNO:1) were each analyzed as described in Example 1. Further analysis ofthe events of UAC-22 indicated that Ceres Clone 1807011 contains adeletion of a nucleotide at position 667 of SEQ ID NO:1, resulting inthe production of a truncated protein. UAC-FA4 and UAC-NK4K were used ascontrols and NREL SWG was used as a standard reference. UAC-FA4 is awild-type plant regenerated from callus. UAC-NB4K corresponds to plantsthat were regenerated from callus transformed with an empty vector(i.e., with no insert). NREL SWG is a composite switchgrass biomassobtained from National Renewable Energy Laboratory (NREL) and was usedas a method control to determine consistency of analytical techniques.The amount of total glucose released per gram dry weight, PLN, and SACvalues are presented in Table 4 for four experiments in which differentamount of enzymes were used in the saccharification analysis. Increasedtotal glucose released per gram of dry weight was observed for each ofthe transgenic lines regardless of the enzyme amount. At standard levelamount of enzymes (i.e., 20 mgP/g), the total glucose released by thetransgenic lines UAC-15-6 and UAC-19-2 was higher than that of thecontrols and the reference standard. This increase was primarily due tothe increase of glucose released during the pretreatment. When theamount of enzymes was reduced by 8-fold (i.e., 2.5 mgP/g), the totalglucose released by the transgenic lines UAC-15-6 and UAC-19-2 wassimilar to the control treated at the standard enzyme level.

Example 8 NIR Conversion Prediction for Transgenic Line PV00460

T₀ tissues from three events of PV00460 containing Ceres Clone 240112(SEQ ID NO:245) were analyzed as described in Example 1. Pv-WT(A26)-72was the wild-type control used, which corresponds to a regenerated butuntransformed plant. The amount of total glucose released per g dryweight, PLN, and SAC values are presented in Table 5 for fourexperiments in which different amount of enzymes were used in thesaccharification analysis. Increased total glucose released per gram ofdry weight was observed for each of the transgenic lines regardless ofthe enzyme amount. At standard level amount of enzymes (i.e., 20 mgP/g),the total glucose released by the transgenic lines PV00460 (especiallyevent #18) was higher than that of the control. This increase wasprimarily due to the increase of glucose released during thepretreatment. When the amount of enzymes was reduced by 8-fold (i.e.,2.5 mgP/g), the total glucose released by the PV00460 transgenic line(for example event #18) was similar to the control treated at thestandard enzyme level.

TABLE 4 Total Glucose Enzymes Released Per g dry Type Lines Amountweight StdDev PLN StdDev SAC stdev Control NREL SWG  20 mgP/g 272.8118.21 45.47 1.86 227.34 16.34 Control UAC-NB4K-1  20 mgP/g 280.54 6.2059.78 0.85 220.77 5.35 Control UAC-FA4-1  20 mgP/g 328.69 23.57 118.383.69 210.30 19.88 Transgenic Line UAC-15-6  20 mgP/g 332.16 11.11 158.014.79 174.15 6.32 Transgenic Line UAC-19-2  20 mgP/g 322.05 7.47 124.063.36 198.00 10.82 Transgenic Line UAC-22-11  20 mgP/g 336.61 12.68113.24 1.67 223.37 14.34 Control NREL SWG 5.0 mgP/g 208.86 13.36 43.821.43 165.04 11.93 Control UAC-NB4K-1 5.0 mgP/g 234.31 16.46 57.90 3.30176.41 13.16 Control UAC-FA4-1 5.0 mgP/g 263.34 4.04 111.18 1.07 152.165.11 Transgenic Line UAC-15-6 5.0 mgP/g 293.04 12.46 154.26 6.15 138.786.32 Transgenic Line UAC-19-2 5.0 mgP/g 276.58 15.36 117.96 4.86 158.6110.51 Transgenic Line UAC-22-11 5.0 mgP/g 287.47 13.08 115.32 0.17172.16 12.91 Control NREL SWG 2.5 mgP/g 184.61 15.02 44.98 3.59 139.6311.42 Control UAC-NB4K-1 2.5 mgP/g 200.15 11.21 61.69 1.82 138.45 9.40Control UAC-FA4-1 2.5 mgP/g 222.18 6.37 114.04 0.67 108.14 7.03Transgenic Line UAC-15-6 2.5 mgP/g 268.36 8.95 155.02 3.25 113.33 5.70Transgenic Line UAC-19-2 2.5 mgP/g 239.20 22.92 121.76 4.39 117.44 18.53Transgenic Line UAC-22-11 2.5 mgP/g 247.62 21.78 115.54 3.83 132.0917.95 Control NREL SWG 1.0 mgP/g 125.35 13.67 41.55 2.81 83.80 10.86Control UAC-NB4K-1 1.0 mgP/g 153.46 5.10 61.19 1.88 92.27 3.22 ControlUAC-FA4-1 1.0 mgP/g 184.70 2.55 119.55 2.57 65.15 0.02 Transgenic LineUAC-15-6 1.0 mgP/g 243.99 1.27 164.69 2.24 79.30 0.97 Transgenic LineUAC-19-2 1.0 mgP/g 203.52 0.86 120.25 4.05 83.27 4.90 Transgenic LineUAC-22-11 1.0 mgP/g 202.32 2.31 115.67 4.54 86.65 6.85

TABLE 5 Total Glucose Enzymes Released Per g dry Type Lines Amountweight StdDev PLN StdDev SAC StdDev Control NREL SWG  20 mgP/g 273.3718.08 42.60 2.69 230.77 15.38 Transgenic Line PV00460-15  20 mgP/g333.23 11.09 99.60 2.55 233.62 8.54 Transgenic Line PV00460-18  20 mgP/g344.73 10.71 115.11 1.73 229.61 8.98 Transgenic Line PV00460-22  20mgP/g 323.11 13.52 88.89 0.05 234.22 13.57 Control Pv-WT (A26)-72  20mgP/g 282.99 10.83 53.96 0.99 229.03 9.84 Control NREL SWG 5.0 mgP/g227.04 30.85 44.53 4.33 182.51 26.52 Transgenic Line PV00460-15 5.0mgP/g 310.22 10.57 103.72 0.89 206.50 11.46 Transgenic Line PV00460-185.0 mgP/g 312.94 14.85 116.72 1.37 196.22 13.48 Transgenic LinePV00460-22 5.0 mgP/g 287.63 11.73 89.26 0.04 198.38 11.69 Control Pv-WT(A26)-72 5.0 mgP/g 243.22 4.08 52.16 1.54 191.06 2.54 Control NREL SWG2.5 mgP/g 177.44 11.91 44.51 2.24 132.93 9.67 Transgenic Line PV00460-152.5 mgP/g 268.84 6.50 104.91 0.10 163.94 6.41 Transgenic Line PV00460-182.5 mgP/g 275.39 3.64 119.88 6.48 155.51 10.12 Transgenic LinePV00460-22 2.5 mgP/g 250.62 8.11 89.65 1.49 160.97 6.62 Control Pv-WT(A26)-72 2.5 mgP/g 220.13 1.31 52.16 1.41 167.97 2.71 Control NREL SWG1.0 mgP/g 122.39 6.33 41.22 2.45 81.17 3.88 Transgenic Line PV00460-151.0 mgP/g 207.42 9.99 106.12 2.86 101.30 7.13 Transgenic Line PV00460-181.0 mgP/g 218.39 3.17 119.80 4.70 98.59 7.87 Transgenic Line PV00460-221.0 mgP/g 196.32 1.90 93.97 1.09 102.35 2.99 Control Pv-WT (A26)-72 1.0mgP/g 141.59 5.82 51.56 0.52 90.03 6.34 Control NREL SWG   0 mgP/g 47.390.91 42.67 0.49 4.72 0.42 Transgenic Line PV00460-15   0 mgP/g 107.200.32 104.09 0.70 3.11 0.39 Transgenic Line PV00460-18   0 mgP/g 119.002.87 116.43 2.45 2.57 0.42 Transgenic Line PV00460-22   0 mgP/g 94.541.21 91.87 1.64 2.67 0.43 Control Pv-WT (A26)-72   0 mgP/g 57.74 0.1154.62 0.07 3.12 0.18

Example 9

Transgenic sorghum plants were made using the same construct containingCeres Cone 1807011 (SEQ ID NO:1) as was used to make the transgenicswitchgrass of Examples 3, 6, and 7. As described above, this results inthe production of a truncated protein (e.g., about 142 residues inlength). Sorghum stalk juice samples were harvested from four eventscontaining Ceres Clone 1807011 and a control plant at approximately softto hard dough stages. After harvesting, the Brix value of each juicesample was measured using a refractometer.

HPLC was carried out with the sorghum juice stalk extracts. Samples wererun on HPLC (Agilent 1100 series) to determine the sugar profile. A HPLCcarbohydrate analysis column (Aminex® HPX-87P column) was used for thesugar analysis. The column was heated at 80° C. and the flow rate wasset at 1 ml/min for analyzing extracts, respectively. Corona® CAD®detector (Thermo Scientific) was used to analyze the sugar samples. Thedata was analyzed using Agilent Chemstation software.

Table 6 presents the Brix and HPLC-determined sugar profiles from juicesamples of transgenic and control plants. The data for each event werebased on one juice sample for single plants. Each sample was divided torun in duplicate so the data represent an average of the duplicates foreach sample. As shown in Table 6, all four transgenic events had anincreased sucrose content, an increased total sugar content, and anincreased Brix value compared to the control event. The sucrose contentranged from 48.18 to 75.85 mg/ml, with two of the events having asucrose content of 62.23 to 75.85 mg/ml. The total sugar content rangedfrom 54.04 to 80.57 mg/ml, with three of the events having a total sugarcontent of 63.13 to 80.57 mg/ml. The Brix value ranged from 10.5 to13.1%, with two of the events having a Brix value that ranged from 11.8to 13.1%. Two of the transgenic events also had an increased glucosecontent compared to the control event.

TABLE 6 Brix Suc Glc Frc Total value Sample name (mg/ml) (mg/ml) (mg/ml)sugars (%) Transgenic 204-02 54.54 5.19 3.4 63.13 10.5 Transgenic 204-1162.23 5.93 4.09 72.25 11.8 Transgenic 204-25 48.18 3.66 2.2 54.04 11Transgenic 204-26 75.85 3.06 1.66 80.57 13.1 204-07 (per 13.78 3.26 2.2219.26 6.6 negative)

Example 10 Determination of Functional Homologs by Reciprocal BLAST

A candidate sequence was considered a functional homolog of a referencesequence if the candidate and reference sequences encoded proteinshaving a similar function and/or activity. A process known as ReciprocalBLAST (Rivera et al., Proc. Natl. Acad. Sci. USA, 95:6239-6244 (1998))was used to identify potential functional homolog sequences fromdatabases consisting of all available public and proprietary peptidesequences, including NR from NCBI and peptide translations from Ceresclones.

Before starting a Reciprocal BLAST process, a specific referencepolypeptide was searched against all peptides from its source speciesusing BLAST in order to identify polypeptides having BLAST sequenceidentity of 80% or greater to the reference polypeptide and an alignmentlength of 85% or greater along the shorter sequence in the alignment.The reference polypeptide and any of the aforementioned identifiedpolypeptides were designated as a cluster.

The BLASTP version 2.0 program from Washington University at SaintLouis, Mo., USA was used to determine BLAST sequence identity andE-value. The BLASTP version 2.0 program includes the followingparameters: 1) an E-value cutoff of 1.0e-5; 2) a word size of 5; and 3)the -postsw option. The BLAST sequence identity was calculated based onthe alignment of the first BLAST HSP (High-scoring Segment Pairs) of theidentified potential functional homolog sequence with a specificreference polypeptide. The number of identically matched residues in theBLAST HSP alignment was divided by the HSP length, and then multipliedby 100 to get the BLAST sequence identity. The HSP length typicallyincluded gaps in the alignment, but in some cases gaps were excluded.

The main Reciprocal BLAST process consists of two rounds of BLASTsearches; forward search and reverse search. In the forward search step,a reference polypeptide sequence, “polypeptide A,” from source speciesSA was BLASTed against all protein sequences from a species of interest.Top hits were determined using an E-value cutoff of 10⁻⁵ and a sequenceidentity cutoff of 35%. Among the top hits, the sequence having thelowest E-value was designated as the best hit, and considered apotential functional homolog or ortholog. Any other top hit that had asequence identity of 80% or greater to the best hit or to the originalreference polypeptide was considered a potential functional homolog orortholog as well. This process was repeated for all species of interest.

In the reverse search round, the top hits identified in the forwardsearch from all species were BLASTed against all protein sequences fromthe source species SA. A top hit from the forward search that returned apolypeptide from the aforementioned cluster as its best hit was alsoconsidered as a potential functional homolog.

Functional homologs were identified by manual inspection of potentialfunctional homolog sequences. Representative functional homologs for SEQID NOs: 483, 562, 246, 111, 348, 774, 416, 2, 157, 280, 641, and 26 areshown in FIGS. 1-12 , respectively. Additional exemplary homologs arecorrelated to certain Figures in the Sequence Listing.

Example 11 Determination of Functional Homologs by Hidden Markov Models

Hidden Markov Models (HMMs) were generated by the program HMMER 2.3.2.To generate each HMM, the default HMMER 2.3.2 program parameters,configured for global alignments, were used.

An HMM was generated using the sequences shown in FIG. 1 as input. Thesesequences were fitted to the model and a representative HMM bit scorefor each sequence is shown in the Sequence Listing. Additional sequenceswere fitted to the model, and representative HMM bit scores for any suchadditional sequences are shown in the Sequence Listing. The resultsindicate that these additional sequences are functional homologs of SEQID NO: 483.

The procedure above was repeated and an HMM was generated for each groupof sequences shown in FIGS. 2-12 , using the sequences shown in eachFigure as input for that HMM. A representative bit score for eachsequence is shown in the Sequence Listing. Additional sequences werefitted to certain HMMs, and representative HMM bit scores for suchadditional sequences are shown in the Sequence Listing. The resultsindicate that these additional sequences are functional homologs of thesequences used to generate that HMM.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of producing a transgenic plant, saidmethod comprising growing a plant cell comprising an exogenous nucleicacid, said exogenous nucleic acid comprising a regulatory regionoperably linked to a heterologous nucleic acid molecule, wherein thenucleic acid molecule encodes a polypeptide having 95 percent or greatersequence identity to the amino acid sequence of SEQ ID NO:416 andwherein the polypeptide comprises a COBRA domain, regenerating thetransgenic plant from the plant cell, selecting the transgenic plant forhaving an increased sucrose content as compared to a control plant thatdoes not comprise said exogenous nucleic acid, and wherein the increasedsucrose content comprises at least 9.66% sucrose of dry biomass of thetransgenic plant.
 2. The method of claim 1, wherein the polypeptidecomprises the amino acid sequence of SEQ ID NO:416.
 3. The method ofclaim 1, wherein the transgenic plant has the increased sucrose content.4. The method of claim 1, wherein the transgenic plant has the increasedglucose release.
 5. The method of claim 1, wherein said nucleic acidmolecule comprises a polynucleotide sequence comprising thepolynucleotide sequence of SEQ ID NO:415.
 6. A method of modulatingbiomass composition in a plant, said method comprising introducing intoa plant an exogenous nucleic acid, said exogenous nucleic acidcomprising a regulatory region operably linked to a heterologous nucleicacid molecule, wherein the nucleic acid molecule encodes a polypeptidehaving 95 percent or greater sequence identity to the amino acidsequence of SEQ ID NO:416 and wherein the polypeptide comprises a COBRAdomain, selecting the plant for having an increased sucrose content ascompared to a plant that does not comprise said nucleic acid, andwherein the increased sucrose content comprises at least 9.66% sucroseof dry biomass of the transgenic plant.
 7. The method of claim 6,wherein the transgenic plant has the increased sucrose content.
 8. Themethod of claim 6, wherein the transgenic plant has the increasedglucose release.
 9. The method of claim 6, wherein said polypeptide has97 percent or greater sequence identity to the amino acid sequence ofSEQ ID NO:416.
 10. The method of claim 6, wherein said nucleic acidmolecule has 97 percent or greater sequence identity to thepolynucleotide sequence of SEQ ID NO:415.
 11. A transgenic plantcomprising an exogenous nucleic acid, said exogenous nucleic acidcomprising: a regulatory region operably linked to a heterologousnucleic acid molecule encoding a polypeptide comprising an amino acidsequence having 95 percent or greater sequence identity to the aminoacid sequence of SEQ ID NO:416 and wherein the polypeptide comprises aCOBRA domain, wherein said transgenic plant is selected for having anincreased sucrose content as compared to a control plant that does notcomprise said nucleic acid, and wherein the increased sucrose contentcomprises at least 9.66% sucrose of dry biomass of the transgenic plant.12. The transgenic plant of claim 11, wherein said plant is a member ofa species selected from the group consisting of Panicum virgatum(switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthusgiganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera(poplar), Zea mays (corn), Glycine max (soybean), Brassica napus(canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryzasativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa),Beta vulgaris (sugarbeet), and Pennisetum glaucum (pearl millet). 13.The transgenic plant of claim 11, wherein said polypeptide comprises theamino acid sequence of SEQ ID NO:416.
 14. A seed product comprisingembryonic tissue from the transgenic plant according to claim 11,wherein the seed product comprises the exogenous nucleic acid.
 15. Acell from the transgenic plant of claim 11, wherein the cell comprisesthe exogenous nucleic acid.